阿可的窩 Yi-Juai

Q: 為何做了 cDNA microarray, 還要做real-time PCR?

(整理自維基 by陳怡睿)
 

Loss of heterozygosity
(節錄自維基 by陳怡睿)
Loss of heterozygosity (LOH) in a cell represents the loss of normal function of one allele of a gene in which the other allele was already inactivated. This term is mostly used in the context of oncogenesis; after an inactivating mutation in one allele of a tumor suppressor gene occurs in the parent's germline cell, it is passed on to the zygote resulting in an offspring that is heterozygous for that allele. In oncology, loss of heterozygosity occurs when the remaining functional allele in a somatic cell of the offspring becomes inactivated by mutation. This results in no normal tumor suppressor being produced and almost certainly results in tumorigenesis.
In cancer
It is a common occurrence in cancer, where it indicates the absence of a functional tumor suppressor gene in the lost region. However, many people remain healthy with such a loss, because there still is one functional gene left on the other chromosome of the chromosome pair. However, the remaining copy of the tumor suppressor gene can be inactivated by a point mutation, leaving no tumor suppressor gene to protect the body.
Retinoblastoma
The classical example of such a loss of protecting genes is hereditary retinoblastoma, in which one parent's contribution of the tumor suppressor Rb1 is flawed. Although most cells will have a functional second copy, chance loss of heterozygosity events in individual cells almost invariably lead to the development of this retinal cancer in the young child.
Detection
Loss of heterozygosity can be identified in cancers by noting the presence of heterozygosity at a genetic locus in an organism's germline DNA, and the absence of heterozygosity at that locus in the cancer cells. This is often done using polymorphic markers, such as microsatellites or single nucleotide polymorphisms, for which the two parents contributed different alleles.

Gene silencing
(節錄自維基 by陳怡睿)
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell.
Genes are regulated at either the transcriptional or post-transcriptional level.
Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.).
Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNAi.
Both transcriptional and post-transcriptional gene silencing are used to regulate endogenous genes. Mechanisms of gene silencing also protect the organism's genome from transposons and viruses. Gene silencing thus may be part of an ancient immune system protecting from such infectious DNA elements.
Genes may be silenced by DNA methylation during meiosis, as in the filamentous fungus Neurospora crassa.

Epigenetics
(節錄自維基 by陳怡睿)
In biology, the term epigenetics refers to heritable changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence. These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. The best example of epigenetic changes in eukaryotic biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells become the various pluripotent cell lines of the embryo which in turn become fully differentiated cells. In other words, a single fertilized egg cell - the zygote - changes into the many cell types including neurons, muscle cells, epithelium, blood vessels et cetera as it continues to divide. It does so by activating some genes while inhibiting others.
Etymology and definitions
Epigenetics (as in "epigenetic landscape") was coined by C. H. Waddington in 1942 as a portmanteau of the words genetics and epigenesis. Epigenesis is an older word to describe the differentiation of cells from their initial totipotent state in embryonic development. When Waddington coined the term the physical nature of genes and their role in heredity was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype.
Robin Holliday defined epigenetics as "the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms." Thus epigenetic can be used to describe any aspect other than DNA sequence that influences the development of an organism.
The modern usage of the word is more narrow, referring to heritable traits (over rounds of cell division and sometimes transgenerationally) that do not involve changes to the underlying DNA sequence. The Greek prefix epi- in epigenetics implies features that are "on top of" or "in addition to" genetics; thus epigenetic traits exist on top of or in addition to the traditional molecular basis for inheritance.
Molecular basis of epigenetics
The molecular basis of epigenetics is complex. It involves modifications of the activation of certain genes, but not the basic structure of DNA. Additionally, the chromatin proteins associated with DNA may be activated or silenced. What this means is that every cell in your body has the same instruction manual, but different cell types are using different chapters. For example, your neurons contain the DNA instructions to make your fingernails, but for these and most other cells in the body, the necessary genes are turned off. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime, but some epigenetic changes are inherited from one generation to the next. Specific epigenetic processes include paramutation, bookmarking, imprinting, gene silencing, X chromosome inactivation, position effect, reprogramming, transvection, maternal effects, the progress of carcinogenesis, many effects of teratogens, regulation of histone modifications and heterochromatin, and technical limitations affecting parthenogenesis and cloning.
Epigenetic research uses a wide range of molecular biologic techniques to further our understanding of epigenetic phenomena, including chromatin immunoprecipitation (together with its large-scale variants ChIP-on-chip and ChIP-seq), fluorescent in situ hybridization, methylation-sensitive restriction enzymes, DNA adenine methyltransferase identification (DamID) and bisulfite sequencing. Furthermore, the use of bioinformatic methods is playing an increasing role (computational epigenetics).
Mechanisms
Several types of epigenetic inheritance systems may play a role in what has become known as cell memory:
DNA methylation and chromatin remodeling
Because the phenotype of a cell or individual is affected by which of its genes are transcribed, heritable transcription states can give rise to epigenetic effects. There are several layers of regulation of gene expression. One way that genes are regulated is through the remodeling of chromatin. Chromatin is the complex of DNA and the histone proteins with which it associates. Histone proteins are little spheres that DNA wraps around. If the way that DNA is wrapped around the histones changes, gene expression can change as well. Chromatin remodeling is initiated by one of two things:
1. The first way is post translational modification of the amino acids that make up histone proteins.
2. The second way is the addition of methyl groups to the DNA, at CpG sites, to convert cytosine to 5-methylcytosine.
The way that the cells stay differentiated in the case of DNA methylation is more clear to us than it is in the case of histone shape. Basically, certain enzymes (such as Dnmt1) "prefer" the methylated cytosine. If this enzyme comes to a "hemimethylated" portion of DNA (DNA where only one strand contains the methylcytosine, and the other side still contains cytosine) the enzyme will methylate the other half.
Although modifications occur throughout the histone sequence, the unstructured termini of histones (called histone tails) are particularly highly modified. These modifications include acetylation, methylation and ubiquitylation. Acetylation is the most highly studied of these modifications. For example, acetylation of the K14 and K9 lysines of the tail of histone H3 by histone acetyltransferase enzymes (HATs) is generally correlated with transcriptional competence.
One mode of thinking is that this tendency of acetylation to be associated with "active" transcription is biophysical in nature. Because lysine normally has a positive charge on the nitrogen at its end, lysine can bind the negatively charged phosphates of the DNA backbone and prevent them from repelling each other. The acetylation event converts the positively charged amine group on the side chain into a neutral amide linkage. This removes the positive charge causing the DNA to repel itself. When this occurs, complexes like SWI/SNF and other transcriptional factors can bind to the DNA, thus opening it up and exposing it to enzymes like RNA polymerase so transcription of the gene can occur.
In addition, the positively charged tails of histone proteins from one nucleosome may interact with the histone proteins on a neighboring nucleosome, causing them to pack closely. Lysine acetylation may interfere with these interactions, causing the chromatin structure to open up.
Lysine acetylation may also act as a beacon to recruit other activating chromatin modifying enzymes (and basal transcription machinery as well). Indeed, the bromodomain—a protein segment (domain) that specifically binds acetyl-lysine—is found in many enzymes that help activate transcription including the SWI/SNF complex (on the protein polybromo). It may be that acetylation acts in this and the previous way to aid in transcriptional activation.
The idea that modifications act as docking modules for related factors is borne out by histone methylation as well. Methylation of lysine 9 of histone H3 has long been associated with constitutively transcriptionally silent chromatin (constitutive heterochromatin). It has been determined that a chromodomain (a domain that specifically binds methyl-lysine) in the transcriptionally repressive protein HP1 recruits HP1 to K9 methylated regions. One example that seems to refute the biophysical model for acetylation is that tri-methylation of histone H3 at lysine 4 is strongly associated with (and required for full) transcriptional activation. Tri-methylation in this case would introduce a fixed positive charge on the tail.
It should be emphasized that differing histone modifications are likely to function in differing ways; acetylation at one position is likely to function differently than acetylation at another position. Also, multiple modifications may occur at the same time, and these modifications may work together to change the behavior of the nucleosome. The idea that multiple dynamic modifications regulate gene transcription in a systematic and reproducible way is called the histone code.
DNA methylation frequently occurs in repeated sequences, and may help to suppress 'junk DNA': Because 5-methylcytosine is chemically very similar to thymidine, CpG sites are frequently mutated and become rare in the genome, except at CpG islands where they remain unmethylated. Epigenetic changes of this type thus have the potential to direct increased frequencies of permanent genetic mutation. DNA methylation patterns are known to be established and modified in response to environmental factors by a complex interplay of at least three independent DNA methyltransferases, DNMT1, DNMT3A and DNMT3B, the loss of any of which is lethal in mice. DNMT1 is the most abundant methyltransferase in somatic cells, localizes to replication foci, has a 10–40-fold preference for hemimethylated DNA and interacts with the proliferating cell nuclear antigen (PCNA). By preferentially modifying hemimethylated DNA, DNMT1 transfers patterns of methylation to a newly synthesized strand after DNA replication, and therefore is often referred to as the ‘maintenance' methyltransferase. DNMT1 is essential for proper embryonic development, imprinting and X-inactivation.
Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of states of larger chromosomal regions are often thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. A simplified stochastic model for this type of epigenetics is found here.
Because DNA methylation and chromatin remodeling play such a central role in many types of epigenic inheritance, the word "epigenetics" is sometimes used as a synonym for these processes. However, this can be misleading. Chromatin remodeling is not always inherited, and not all epigenetic inheritance involves chromatin remodeling.
It has been suggested that the histone code could be mediated by the effect of small RNAs. The recent discovery and characterization of a vast array of small (21- to 26-nt), non-coding RNAs suggests that there is an RNA component, possibly involved in epigenetic gene regulation. Small interfering RNAs can modulate transcriptional gene expression via epigenetic modulation of targeted promoters.
RNA transcripts and their encoded proteins
Sometimes a gene, after being turned on, transcribes a product that (either directly or indirectly) maintains the activity of that gene. For example, Hnf4 and MyoD enhance the transcription of many liver- and muscle-specific genes, respectively, including their own, through the transcription factor activity of the proteins they encode. Other epigenetic changes are mediated by the production of different splice forms of RNA, or by formation of double-stranded RNA (RNAi). Descendants of the cell in which the gene was turned on will inherit this activity, even if the original stimulus for gene-activation is no longer present. These genes are most often turned on or off by signal transduction, although in some systems where syncytia or gap junctions are important, RNA may spread directly to other cells or nuclei by diffusion. A large amount of RNA and protein is contributed to the zygote by the mother during oogenesis or via nurse cells, resulting in maternal effect phenotypes. A smaller quantity of sperm RNA is transmitted from the father, but there is recent evidence that this epigenetic information can lead to visible changes in several generations of offspring.
Prions
Prions are infectious forms of proteins. Proteins generally fold into discrete units which perform distinct cellular functions, but some proteins are also capable of forming an infectious conformational state known as a prion. Although often viewed in the context of infectious disease, prions are more loosely defined by their ability to catalytically convert other native state versions of the same protein to an infectious conformational state. It is in this latter sense that they can be viewed as epigenetic agents capable of inducing a phenotypic change without a modification of the genome.
Fungal prions are considered epigenetic because the infectious phenotype caused by the prion can be inherited without modification of the genome. PSI+ and URE3, discovered in yeast in 1965 and 1971, are the two best studied of this type of prion. Prions can have a phenotypic effect through the sequestration of protein in aggregates, thereby reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in termination of translation) causes ribosomes to have a higher rate of read-through of stop codons, an effect which results in suppression of nonsense mutations in other genes. The ability of Sup35 to form prions may be a conserved trait. It could confer an adaptive advantage by giving cells the ability to switch into a PSI+ state and express dormant genetic features normally terminated by premature stop codon mutations.
Structural inheritance systems
 
In ciliates such as Tetrahymena and Paramecium, genetically identical cells show heritable differences in the patterns of ciliary rows on their cell surface. Experimentally altered patterns can be transmitted to daughter cells. It seems existing structures act as templates for new structures. The mechanisms of such inheritance are unclear, but reasons exist to assume that multicellular organisms also use existing cell structures to assemble new ones.[28]
Functions and consequences
Development
Somatic epigenetic inheritance, particularly through DNA methylation and chromatin remodeling, is very important in the development of multicellular eukaryotic organisms. The genome sequence is static (with some notable exceptions), but cells differentiate in many different types, which perform different functions, and respond differently to the environment and intercellular signalling. Thus, as individuals develop, morphogens activate or silence genes in an epigenetically heritable fashion, giving cells a "memory". In mammals, most cells terminally differentiate, with only stem cells retaining the ability to differentiate into several cell types ("totipotency" and "multipotency"). In mammals, some stem cells continue producing new differentiated cells throughout life, but mammals are not able to respond to loss of some tissues, for example, the inability to regenerate limbs, which some other animals are capable of. Unlike animals, plant cells do not terminally differentiate, remaining totipotent with the ability to give rise to a new individual plant. While plants do utilise many of the same epigenetic mechanisms as animals, such as chromatin remodeling, it has been hypothesised that plant cells do not have "memories", resetting their gene expression patterns at each cell division using positional information from the environment and surrounding cells to determine their fate.
Medicine
Epigenetics has many and varied potential medical applications. Congenital genetic disease is well understood, and it is also clear that epigenetics can play a role, for example, in the case of Angelman syndrome and Prader-Willi syndrome. These are normal genetic diseases caused by gene deletions, but are unusually common because individuals are essentially hemizygous because of genomic imprinting, and therefore a single gene knock out is sufficient to cause the disease, where most cases would require both copies to be knocked out.
Evolution
Although epigenetics in multicellular organisms is generally thought to be a mechanism involved in differentiation, with epigenetic patterns "reset" when organisms reproduce, there have been some observations of transgenerational epigenetic inheritance (e.g., the phenomenon of paramutation observed in maize). Although most of these multigenerational epigenetic traits are gradually lost over several generations, the possibility remains that multigenerational epigenetics could be another aspect to evolution and adaptation. These effects may require enhancements to the standard conceptual framework of the modern evolutionary synthesis.
Epigenetic features may play a role in short-term adaptation of species by allowing for reversible phenotype variability. The modification of epigenetic features associated with a region of DNA allows organisms, on a multigenerational time scale, to switch between phenotypes that express and repress that particular gene. Whereas the DNA sequence of the region is not mutated, this change is reversible. It has also been speculated that organisms may take advantage of differential mutation rates associated with epigenetic features to control the mutation rates of particular genes.
Epigenetic changes have also been observed to occur in response to environmental exposure—for example, mice given some dietary supplements have epigenetic changes affecting expression of the agouti gene, which affects their fur color, weight, and propensity to develop cancer.
Epigenetic effects in humans
Genomic imprinting and related disorders
Some human disorders are associated with genomic imprinting, a phenomenon in mammals where the father and mother contribute different epigenetic patterns for specific genomic loci in their germ cells. The most well-known case of imprinting in human disorders is that of Angelman syndrome and Prader-Willi syndrome—both can be produced by the same genetic mutation, chromosome 15q partial deletion, and the particular syndrome that will develop depends on whether the mutation is inherited from the child's mother or from their father. This is due to the presence of genomic imprinting in the region. Beckwith-Wiedemann syndrome is also associated with genomic imprinting, often caused by abnormalities in maternal genomic imprinting of a region on chromosome 11.
Transgenerational epigenetic observations
Marcus Pembrey and colleagues also observed that the paternal (but not maternal) grandsons of Swedish boys who were exposed to famine in the 19th century were less likely to die of cardiovascular disease; if food was plentiful then diabetes mortality in the grandchildren increased, suggesting that this was a transgenerational epigenetic inheritance.
Cancer and developmental abnormalities
A variety of compounds are considered as epigenetic carcinogens—they result in an increased incidence of tumors, but they do not show mutagen activity (toxic compounds or pathogens that cause tumors incident to increased regeneration should also be excluded). Examples include diethylstilbestrol, arsenite, hexachlorobenzene, and nickel compounds.
Many teratogens exert specific effects on the fetus by epigenetic mechanisms. While epigenetic effects may preserve the effect of a teratogen such as diethylstilbestrol throughout the life of an affected child, the possibility of birth defects resulting from exposure of fathers or in second and succeeding generations of offspring has generally been rejected on theoretical grounds and for lack of evidence. However, a range of male-mediated abnormalities have been demonstrated, and more are likely to exist. FDA label information for Vidaza(tm), a formulation of 5-azacitidine (an unmethylatable analog of cytidine that causes hypomethylation when incorporated into DNA) states that "men should be advised not to father a child" while using the drug, citing evidence in treated male mice of reduced fertility, increased embryo loss, and abnormal embryo development. In rats, endocrine differences were observed in offspring of males exposed to morphine. In mice, second generation effects of diethylstilbesterol have been described occurring by epigenetic mechanisms.
Epigenetics in microorganisms
Bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Bacteria make use of DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation is important in bacteria virulence in organisms such as Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage, transposase activity and regulation of gene expression.
The yeast prion PSI is generated by a conformational change of a translation termination factor, which is then inherited by daughter cells. This can provide a survival advantage under adverse conditions. This is an example of epigenetic regulation enabling unicellular organisms to respond rapidly to environmental stress. Prions can be viewed as epigenetic agents capable of inducing a phenotypic change without modification of the genome.

DNA methylation
(節錄自維基 by陳怡睿)
DNA methylation is a type of chemical modification of DNA that can be inherited and subsequently removed without changing the original DNA sequence. As such, it is part of the epigenetic code and is also the most well characterized epigenetic mechanism.
DNA methylation involves the addition of a methyl group to DNA — for example, to the number 5 carbon of the cytosine pyrimidine ring — in this case with the specific effect of reducing gene expression. DNA methylation at the 5 position of cytosine has been found in every vertebrate examined. In adult somatic tissues, DNA methylation typically occurs in a CpG dinucleotide context; non-CpG methylation is prevalent in embryonic stem cells.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and asymmetrically (CpNpNp), where N can be any nucleotide but guanine. Some organisms, such as fruit flies, exhibit virtually no DNA methylation.
Research has suggested that long term memories in humans may be stored via DNA methylation.
In mammals
DNA methylation is essential for normal development and is associated with a number of key processes including imprinting, X-chromosome inactivation, suppression of repetitive elements and carcinogenesis.
Between 60-90% of all CpGs are methylated in mammals. Unmethylated CpGs are grouped in clusters called "CpG islands" that are present in the 5' regulatory regions of many genes. In many disease processes such as cancer, gene promoter CpG islands acquire abnormal hypermethylation, which results in heritable transcriptional silencing. DNA methylation may impact the transcription of genes in two ways. First, the methylation of DNA may itself physically impede the binding of transcriptional proteins to the gene and secondly, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodelling proteins that can modify histones, thereby forming compact, inactive chromatin termed silent chromatin. This link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has been implicated in Rett syndrome and methyl-CpG binding domain protein 2 (MBD2) mediates the transcriptional silencing of hypermethylated genes in cancer.
DNA Methyltransferases
In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleodtides and carried out by two general classes of enzymatic activities - maintenance methylation and de novo methylation.
Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the DNA methyltransferase, the replication machinery itself would produce daughter strands that are unmethylated and overtime would lead to passive demethylation. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.
It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 (TRDMT1) has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.
Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-aza-2'-deoxycytidine (decitabine) is a nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, but this can cause mutations in the daughter cells if the cell does not die. Additionally, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation. However, it is currently unclear if targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.
In plants
Significant progress has been made in understanding DNA methylation in plants, specifically in the model plant, Arabidopsis thaliana. Whereas in mammals methylation mainly occurs on the cytosine in a CpG context, in plants the cytosine can be methylated in the CpG, CpNpG, and CpNpN context, where N represents any nucleotide but guanine.
The principal Arabidopsis DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 protein is unique to the plant kingdom. There are currently two classes of DNA methyltransferases: 1) the de-novo class, or enzymes that create new methylation marks on the DNA, and 2) a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughters strands after DNA replication. DRM2 is the only enzyme that has been implicated as a de-novo DNA methyltransferase. DRM2 has also been shown, along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication. Currently, it is not clear how the cell determines the locations of de-novo DNA methylation, but evidence suggests that for many, though not all locations, RNA-directed DNA methylation (RdDM) is involved. In RdDM, specific RNA transcripts are produced from a genomic DNA template, and this RNA forms secondary structures called double-stranded RNA molecules. The double-stranded RNAs, through either the small interfering RNA (siRNA) or microRNA (miRNA) pathways, direct de-novo DNA methylation of the original genomic location that produced the RNA. This sort of mechanism is thought to be important in cellular defense against RNA viruses and/or transposons both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, through a still-poorly-understood mechanism, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect.
In fungi
Many fungi apparently have low levels (0.1 to 0.5%) of cytosine methylation while other fungi have as much as 5% of the genome methylated. This value seems to vary both among species and among isolates of the same species. There is also evidence that DNA methylation may be involved in state-specific control of gene expression in fungi.
Although brewers yeast (Saccharomyces) and fission yeast (Schizosaccharomyces) have very little DNA methylation, the model filamentous fungus Neurospora crassa has a well characterized methylation system. Several genes control methylation in Neurospora and mutation of the DNA methyl transferase, dim-2, eliminates all DNA methylation in Neurospora but does not affect growth or sexual reproduction. While the Neurospora genome has very little repeated DNA, half of the methylation occurs in repeated DNA including transposon relics and centromeric DNA.The ability to evaluate other important phenomena in a DNA methylase deficient genetic background makes Neurospora and important system in which to study DNA methylation.
In bacteria
Adenine or cytosine methylation is part of the restriction modification system of many bacteria, in which DNAs are methylated periodically throughout the genome. A methylase is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzymes. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage. These restriction enzymes are the basis of restriction fragment length polymorphism (RFLP) testing, used to detect DNA polymorphisms.

資料來源: 維基百科 整理: 陳怡睿
ELISA
    Enzyme-Linked ImmunoSorbent Assay, also called ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
    Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.
Applications
    Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
    The ELISA test was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.
One method of determining a cut-off point is by comparison with a known standard. For example, if an ELISA test will be used in workplace drug screening, a cut-off concentration (e.g., 50 ng/mL of drug) will be established and a sample will be prepared that contains that concentration of analyte. Unknowns that generate a signal that is stronger than the known sample are called "positive"; those that generate weaker signal are called "negative."
Types
"Indirect" ELISA
The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
    The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.
    ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
Sandwich ELISA
    A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
Prepare a surface to which a known quantity of capture antibody is bound.
Block any non specific binding sites on the surface.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply primary antibodies that bind specifically to the antigen.
Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
    The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
Competitive ELISA
    A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
Unlabeled antibody is incubated in the presence of its antigen.
These bound antibody/antigen complexes are then added to an antigen coated well.
The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
    For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal.
ELISA Reverse method & device (ELISA-R m&d)
    A newer technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.
Advantages:
The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays;
The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
One ogive is left unsensitized to measure the non-specific reactions of the sample;
The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.
Radioimmunoassay
    Radioimmunoassay (RIA) is a scientific method used to test antigens (for example, hormone levels in the blood) without the need to use a bioassay. It was developed by Rosalyn Yalow and Solomon Aaron Berson in the 1950s. In 1977, Rosalyn Sussman Yalow received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amounts of such a hormone was considered a breakthrough in endocrinology.
    Although the RIA technique is extremely sensitive and extremely specific, it requires a sophisticated apparatus and is costly. It also requires special precautions, since radioactive substances are used. Therefore, today it has been largely supplanted by the ELISA method, where the antigen-antibody reaction is measured using colorometric signals instead of a radioactive signal.
    To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen for antibody binding sites.
    As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured. A binding curve can then be plotted, and the exact amount of antigen in the patient's serum can be determined.
    With this technique, separating bound from unbound antigen is crucial. Initially, the method of separation employed was the use of a second "anti-antibody" directed against the first for precipitation and centrifugation. The use of charcoal suspension for precipitation was extended but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4. An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.
Applications
    The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., blood banking, diagnosis of allergies,  and endocrinology.

ELISA (包括western blot及其比較與應用)

ELISA
Enzyme-Linked ImmunoSorbent Assay, also called ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.
A 96-well microtiter plate being used for ELISA.
Method
The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used. The test requires:
* the antibodies fixed to a solid surface, such as the inner surface of a test tube;
* a preparation of the same antibodies coupled to an enzyme. This is one (e.g., β-galactosidase) that produces a colored product from a colorless substrate.
Performing the Test
1. The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules.
2. The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich".
 3. After washing away any unbound conjugate, the substrate solution is added.
 4. After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.
ELISA can also be adapted to measure the concentration of antibodies. In this case,
1. The wells are coated with the appropriate antigen.
2. The solution (e.g., serum) containing antibodies is added.
3. After they have had time to bind to the immobilized antigen,
4. an enzyme-conjugated anti-immunoglobulin is added, consisting of an antibody against the antibodies being tested for. For example, if human anti-HIV antibodies are being assayed, then antibodies (raised in a goat or rabbit against human immunoglobulins) are conjugated to the enzyme.
5. After washing away unreacted reagent, the substrate is added.
6. The intensity of the color produced is proportional to the amount of enzyme-labeled antibodies bound (and thus to the concentration of the antibodies being assayed).
Types
"Indirect" ELISA
The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
1. Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
2. A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
3. The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
4. The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
5. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
6. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
8. View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
Sandwich ELISA
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is bound.
2. Block any non specific binding sites on the surface.
3. Apply the antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply primary antibodies that bind specifically to the antigen.
6. Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
7. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
8. Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
9. Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen.
2. These bound antibody/antigen complexes are then added to an antigen coated well.
3. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal).
ELISA Reverse method & device (ELISA-R m&d)
A newer technique uses a solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.
Advantages:
The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays;
The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples;
3. One ogive is left unsensitized to measure the non-specific reactions of the sample;
4. The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.
Applications
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.
One method of determining a cut-off point is by comparison with a known standard. For example, if an ELISA test will be used in workplace drug screening, a cut-off concentration (e.g., 50 ng/mL of drug) will be established and a sample will be prepared that contains that concentration of analyte. Unknowns that generate a signal that is stronger than the known sample are called "positive"; those that generate weaker signal are called "negative."
Some examples:
    * screening donated blood for evidence of viral contamination by
          o HIV-1 and HIV-2 (presence of anti-HIV antibodies)
          o hepatitis C (presence of antibodies)
          o hepatitis B (testing for both antibodies and a viral antigen)
          o HTLV-1 and -2 (presence of antibodies)
    * measuring hormone levels
          o HCG (as a test for pregnancy)
          o LH (determining the time of ovulation)
          o TSH, T3 and T4 (for thyroid function)
          o hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes
    * detecting infections
          o sexually-transmitted agents like HIV, syphilis, and chlamydia
          o hepatitis B and C
          o Toxoplasma gondii
    * detecting allergens in food and house dust
    * measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like lupus erythematosus
    * measuring toxins in contaminated food
    * detecting illicit drugs, e.g.,
          o cocaine
          o opiates
          o Δ-9-tetrahydrocannabinol, the active ingredient in marijuana
摘要 ELISA (Enzyme-Linked ImmunoSorbant Assay)
The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much. There are two main variations on this method: you can determine how much antibody is in a sample, or you can determine how much protein is bound by an antibody. The distinction is whether you are trying to quantify an antibody or some other protein. In this example, we will use an ELISA to determine how much of a particular antibody is present in an individuals blood.
ELISAs are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with a protein to which will bind the antibody you want to measure. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies (called primary antibodies). The serum is incubated in a well, and each well contains a different serum (see figure below). A positive control serum and a negative control serum would be included among the 96 samples being tested.
After some time, the serum is removed and weakly adherent antibodies are washed off with a series of buffer rinses. To detect the bound antibodies, a secondary antibody is added to each well. The secondary antibody would bind to all human antibodies and is typically produced in a rodent. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products. After an incubation period, the secondary antibody solution is removed and loosely adherent ones are washed off as before. The final step is the addition the enzyme substrate and the production of colored product in wells with secondary antibodies bound.
When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical density (i.e. the amount of colored product) is determined for each well. The amount of color produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells.

# Western blot
The western blot (alternatively, immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.
Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).
The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.
Steps in a Western blot
Tissue preparation
Gel electrophoresis
Immunoblot (Western blot) analysis of proteins separated by SDS-PAGE gradientgel electrophoresis.
The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilo Daltons, kDa). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.
Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.
It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
Transfer
Blocking
Detection
During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications.
Analysis
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is indexed to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
Colorimetric detection
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry.
Chemiluminescent detection
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which captures a digital image of the western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. So-called "enhanced chemiluminescent" (ECL) detection is considered to be among the most sensitive detection methods for blotting analysis.
Radioactive detection
Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest (see image to the right). The importance of radioactive detections methods is declining[citation needed], because it is very expensive, health and safety risks are high and ECL provides a useful alternative.
Fluorescent detection
The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.
Secondary probing
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping, and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use.
2-D Gel Electrophoresis
2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, in the first dimension polypeptides are separated according to isoelectric point, while in the second dimension polypeptides are separated according to their molecular weight. The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine and arginine) and negatively (e.g. glutamate and aspartate) charged amino acids, with negatively charged amino acids contributing to a high isoelectric point and positively charged amino acids contributing to a low isoelectric point. Samples could also be separated first under nonreducing conditions using SDS-PAGE and under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.
In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein - e.g. a protein that has been phosphorylated (by addition of a negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry, which identifies the protein.
Medical diagnostic applications
    * The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.
    * A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').
    * Some forms of Lyme disease testing employ Western blotting.
Western Blot vs. ELISA
Sensitivity & Specificity Differences
    Western blot is very rarely acceptable for detection of HCPs in your drug substance or drug product samples. Samples downstream in your purification process typically contain HCPs below the sensitivity of western blot. For western blot, you are limited in the amount of total protein you can load and still get good PAGE resolution. When you load final product or samples from downstream in the purification process the vast majority of protein will be the product itself. For example, the maximal load of protein for a PAGE run on a mini gel is on the order of 10 μg/lane. If HCP contamination is 100 ppm, a level typical of many final drug products, then the amount of total HCP in that 10μg of drug would be 1 ng. With the sensitivity of western blot on the order of 1 ng/band it could in theory detect HCP contamination down to 100 ppm if the 100 ppm were a single HCP and not a mixture of several different HCPs. As it turns out there are usually several HCPs that contaminate final product and for this reason western blot is almost always negative for HCP on downstream and final product samples. ELISA demonstrates less interference from drug product and shows sensitivity more than 100 fold lower than western blot. As such, ELISA will typically allow for the detection of total HCP contamination to less than 1 ppm. There are many other fundamental reasons why the sensitivity of western blot is inferior to ELISA.
    For example, western blot often requires that the PAGE step be carried out under reducing conditions (DTT or BME followed by boiling) and in the presence of high concentrations of SDS detergent. These procedural components may actually denature or block some of the native HCP epitopes that would be detectable in an ELISA. Incomplete transfer of the proteins out of the PAGE and onto the membrane and adsorption on the membrane at or near antigenic sites will also limit the amount of binding seen by western blot. As you try to increase the sensitivity of western blot it is very common that the specificity of the method is also compromised. What is typically seen is that a non-immunoreactive protein present in very high concentration (e.g. your drug substance) will invariably adsorb some of the excess anti-HCP antibody nonspecifically leading to the erroneous conclusion that the anti-HCP antibody seems to "cross react" with your product. The way to confirm this non-specific binding to your product is to use a non-immune immunoglobulin of the same species and at the same concentration as the anti-HCP antibody. If the intensity of the drug substance band is the same with both the normal goat IgG and the anti-HCP antibody, you can conclude the band is non-specific. Beyond that experiment it should be understood that the specificity of the ELISA method is typically orders of magnitude better than western blot owing in large part to the fact that any protein must be bound simultaneously by both the capture antibody and the detection antibody. For this reason most artifactual product bands in the western will not yield apparent HCP activity in the ELISA method.
ELISA/Western blot tests for HIV
(Update Date: 1/22/2008 Updated by: Kenneth M. Wener, MD, Department of Infectious Diseases, Lahey Clinic, Burlington, MA. Review provided by VeriMed Healthcare Network. A.D.A.M.)

主題一: Stem Cell的定義
摘錄自: Stem Cell Information (The official National Institutes of Health resource for stem cell research)
I. What are stem cells and why are they important?
Stem cells have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods through cell division. The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulinproducing cells of the pancreas.
Scientists primarily work with two kinds of stem cells from animals and humans: embryonic stem cells and adult stem cells, which have different functions and characteristics that will be explained in this document. Scientists discovered ways to obtain or derive stem cells from early mouse embryos more than 20 years ago. Many years of detailed study of the biology of mouse stem cells led to the discovery, in 1998, of how to isolate stem cells from human embryos and grow the cells in the laboratory. These are called human embryonic stem cells. The embryos used in these studies were created for infertility purposes through in vitro fertilization procedures and when they were no longer needed for that purpose, they were donated for research with the informed consent of the donor.
Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, stem cells in developing tissues give rise to the multiple specialized cell types that make up the heart, lung, skin, and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury, or disease.
It has been hypothesized by scientists that stem cells may, at some point in the future, become the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease.
Scientists want to study stem cells in the laboratory so they can learn about their essential properties and what makes them different from specialized cell types. As scientists learn more about stem cells, it may become possible to use the cells not just in cell-based therapies, but also for screening new drugs and toxins and understanding birth defects. However, as mentioned above, human embryonic stem cells have only been studied since 1998. Therefore, in order to develop such treatments scientists are intensively studying the fundamental properties of stem cells, which include: 1. determining precisely how stem cells remain unspecialized and self renewing for many years; and 2. identifying the signals that cause stem cells to become specialized cells.
II. What are the unique properties of all stem cells?
Stem cells differ from other kinds of cells in the body. All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.
Scientists are trying to understand two fundamental properties of stem cells that relate to their longterm self-renewal: 1. why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most adult stem cells cannot; and 2. what are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?
Discovering the answers to these questions may make it possible to understand how cell proliferation is regulated during normal embryonic development or during the abnormal cell division that leads to cancer. Importantly, such information would enable scientists to grow embryonic and adult stem cells more efficiently in the laboratory.
Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. A stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell); it cannot carry molecules of oxygen through the bloodstream (like a red blood cell); and it cannot fire electrochemical signals to other cells that allow the body to move or speak (like a nerve cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.
Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells—which do not normally replicate themselves—stem cells may replicate many times. When cells replicate themselves many times over it is called proliferation. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.
The specific factors and conditions that allow stem cells to remain unspecialized are of great interest to scientists. It has taken scientists many years of trial and error to learn to grow stem cells in the laboratory without them spontaneously differentiating into specific cell types. For example, it took 20 years to learn how to grow human embryonic stem cells in the laboratory following the development of conditions for growing mouse stem cells. Therefore, an important area of research is understanding the signals in a mature organism that cause a stem cell population to proliferate and remain unspecialized until the cells are needed for repair of a specific tissue. Such information is critical for scientists to be able to grow large numbers of unspecialized stem cells in the laboratory for further experimentation.
Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. Scientists are just beginning to understand the signals inside and outside cells that trigger stem cell differentiation. The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment.
Therefore, many questions about stem cell differentiation remain. For example, are the internal and external signals for cell differentiation similar for all kinds of stem cells? Can specific sets of signals be identified that promote differentiation into specific cell types? Addressing these questions is critical because the answers may lead scientists to find new ways of controlling stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes including cell-based therapies.
Adult stem cells typically generate the cell types of the tissue in which they reside. A blood-forming adult stem cell in the bone marrow, for example, normally gives rise to the many types of blood cells such as red blood cells, white blood cells and platelets. Until recently, it had been thought that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—could not give rise to the cells of a very different tissue, such as nerve cells in the brain. However, a number of experiments over the last several years have raised the possibility that stem cells from one tissue may be able to give rise to cell types of a completely different tissue, a phenomenon known as plasticity. Examples of such plasticity include blood cells becoming neurons, liver cells that can be made to produce insulin, and hematopoietic stem cells that can develop into heart muscle. Therefore, exploring the possibility of using adult stem cells for cell-based therapies has become a very active area of investigation by researchers.
III. What are embryonic stem cells?
A. What stages of early embryonic development are important for generating embryonic stem cells?
Embryonic stem cells, as their name suggests, are derived from embryos. Specifically, embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro—in an in vitro fertilization clinic—and then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocyst; the blastocoel, which is the hollow cavity inside the blastocyst; and the inner cell mass, which is a group of approximately 30 cells at one end of the blastocoel.
B. How are embryonic stem cells grown in the laboratory?
Growing cells in the laboratory is known as cell culture. Human embryonic stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The reason for having the mouse cells in the bottom of the culture dish is to give the inner cell mass cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Recently, scientists have begun to devise ways of growing embryonic stem cells without the mouse feeder cells. This is a significant scientific advancement because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
Over the course of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called subculturing. Each cycle of subculturing the cells is referred to as a passage. After six months or more, the original 30 cells of the inner cell mass yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line. Once cell lines are established, or even before that stage, batches of them can be frozen and shipped to other laboratories for further culture and experimentation.
C. What laboratory tests are used to identify embryonic stem cells?
At various points during the process of generating embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells. This process is called characterization.
As yet, scientists who study human embryonic stem cells have not agreed on a standard battery of tests that measure the cells' fundamental properties. Also, scientists acknowledge that many of the tests they do use may not be good indicators of the cells' most important biological properties and functions. Nevertheless, laboratories that grow human embryonic stem cell lines use several kinds of tests. These tests include:
Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term self-renewal. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated.
Using specific techniques to determine the presence of surface markers that are found only on undifferentiated cells. Another important test is for the presence of a protein called Oct-4, which undifferentiated cells typically make. Oct-4 is a transcription factor, meaning that it helps turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development.
Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. It does not detect genetic mutations in the cells.
Determining whether the cells can be subcultured after freezing, thawing, and replating.
Testing whether the human embryonic stem cells are pluripotent by 1) allowing the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so they will differentiate to form specific cell types; or 3) injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types—an indication that the embryonic stem cells are capable of differentiating into multiple cell types.
D. How are embryonic stem cells stimulated to differentiate?
As long as the embryonic stem cells in culture are grown under certain conditions, they can remain undifferentiated (unspecialized). But if cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously. They can form muscle cells, nerve cells, and many other cell types. Although spontaneous differentiation is a good indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce cultures of specific cell types.
So, to generate cultures of specific types of differentiated cells—heart muscle cells, blood cells, or nerve cells, for example—scientists try to control the differentiation of embryonic stem cells. They change the chemical composition of the culture medium, alter the surface of the culture dish, or modify the cells by inserting specific genes. Through years of experimentation scientists have established some basic protocols or "recipes" for the directed differentiation of embryonic stem cells into some specific cell types.
If scientists can reliably direct the differentiation of embryonic stem cells into specific cell types, they may be able to use the resulting, differentiated cells to treat certain diseases at some point in the future. Diseases that might be treated by transplanting cells generated from human embryonic stem cells include Parkinson's disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's muscular dystrophy, heart disease, and vision and hearing loss.
Stem Cell Basics
IV. What are adult stem cells?
An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ.
The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Some scientists now use the term somatic stem cell instead of adult stem cell.
Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown.
Research on adult stem cells has recently generated a great deal of excitement. Scientists have found adult stem cells in many more tissues than they once thought possible. This finding has led scientists to ask whether adult stem cells could be used for transplants. In fact, adult blood forming stem cells from bone marrow have been used in transplants for 30 years. Certain kinds of adult stem cells seem to have the ability to differentiate into a number of different cell types, given the right conditions. If this differentiation of adult stem cells can be controlled in the laboratory, these cells may become the basis of therapies for many serious common diseases.
The history of research on adult stem cells began about 40 years ago. In the 1960s, researchers discovered that the bone marrow contains at least two kinds of stem cells. One population, called hematopoietic stem cells, forms all the types of blood cells in the body. A second population, called bone marrow stromal cells, was discovered a few years later. Stromal cells are a mixed cell population that generates bone, cartilage, fat, and fibrous connective tissue.
Also in the 1960s, scientists who were studying rats discovered two regions of the brain that contained dividing cells, which become nerve cells. Despite these reports, most scientists believed that new nerve cells could not be generated in the adult brain. It was not until the 1990s that scientists agreed that the adult brain does contain stem cells that are able to generate the brain's three major cell types—astrocytes and oligodendrocytes, which are non-neuronal cells, and neurons, or nerve cells.
A. Where are adult stem cells found and what do they normally do?
Adult stem cells have been identified in many organs and tissues. One important point to understand about adult stem cells is that there are a very small number of stem cells in each tissue. Stem cells are thought to reside in a specific area of each tissue where they may remain quiescent (non-dividing) for many years until they are activated by disease or tissue injury. The adult tissues reported to contain stem cells include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin and liver.
Scientists in many laboratories are trying to find ways to grow adult stem cells in cell culture and manipulate them to generate specific cell types so they can be used to treat injury or disease. Some examples of potential treatments include replacing the dopamine-producing cells in the brains of Parkinson's patients, developing insulin-producing cells for type I diabetes and repairing damaged heart muscle following a heart attack with cardiac muscle cells.
B. What tests are used for identifying adult stem cells?
Scientists do not agree on the criteria that should be used to identify and test adult stem cells.
However, they often use one or more of the following three methods: (1) labeling the cells in a living tissue with molecular markers and then determining the specialized cell types they generate; (2) removing the cells from a living animal, labeling them in cell culture, and transplanting them back into another animal to determine whether the cells repopulate their tissue of origin; and (3) isolating the cells, growing them in cell culture, and manipulating them, often by adding growth factors or introducing new genes, to determine what differentiated cells types they can become.
Also, a single adult stem cell should be able to generate a line of genetically identical cells—known as a clone—which then gives rise to all the appropriate differentiated cell types of the tissue. a clone—which then gives rise to all the appropriate differentiated cell types of the tissue. Scientists tend to show either that a stem cell can give rise to a clone of cells in cell culture, or that a purified population of candidate stem cells can repopulate the tissue after transplant into an animal. Recently, by infecting adult stem cells with a virus that gives a unique identifier to each individual cell, scientists have been able to demonstrate that individual adult stem cell clones have the ability to repopulate injured tissues in a living animal.
C. What is known about adult stem cell differentiation?
As indicated above, scientists have reported that adult stem cells occur in many tissues and that they enter normal differentiation pathways to form the specialized cell types of the tissue in which they reside. Adult stem cells may also exhibit the ability to form specialized cell types of other tissues, which is known as transdifferentiation or plasticity.
Normal differentiation pathways of adult stem cells. In a living animal, adult stem cells can divide for a long period and can give rise to mature cell types that have characteristic shapes and specialized structures and functions of a particular tissue. The following are examples of differentiation pathways of adult stem cells.
Hematopoietic stem cells give rise to all the types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets.
Bone marrow stromal cells (mesenchymal stem cells) give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons.
Neural stem cells in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal cells—astrocytes and oligodendrocytes.
Epithelial stem cells in the lining of the digestive tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells.
Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.
Adult stem cell plasticity and transdifferentiation. A number of experiments have suggested that certain adult stem cell types are pluripotent. This ability to differentiate into multiple cell types is called plasticity stem cell types are pluripotent. This ability to differentiate into multiple cell types is called plasticity or transdifferentiation. The following list offers examples of adult stem cell plasticity that have been reported during the past few years.
Hematopoietic stem cells may differentiate into: three major types of brain cells (neurons, oligodendrocytes, and astrocytes); skeletal muscle cells; cardiac muscle cells; and liver cells.
Bone marrow stromal cells may differentiate into: cardiac muscle cells and skeletal muscle cells.
Brain stem cells may differentiate into: blood cells and skeletal muscle cells.
Current research is aimed at determining the mechanisms that underlie adult stem cell plasticity. If such mechanisms can be identified and controlled, existing stem cells from a healthy tissue might be induced to repopulate and repair a diseased tissue.
D. What are the key questions about adult stem cells?
Many important questions about adult stem cells remain to be answered. They include:
How many kinds of adult stem cells exist, and in which tissues do they exist?
What are the sources of adult stem cells in the body? Are they "leftover" embryonic stem cells, or do they arise in some other way? Why do they remain in an undifferentiated state when all the cells around them have differentiated?
Do adult stem cells normally exhibit plasticity, or do they only transdifferentiate when scientists manipulate them experimentally? What are the signals that regulate the proliferation and differentiation of stem cells that demonstrate plasticity?
Is it possible to manipulate adult stem cells to enhance their proliferation so that sufficient tissue for transplants can be produced?
Does a single type of stem cell exist—possibly in the bone marrow or circulating in the blood—that can generate the cells of any organ or tissue?
What are the factors that stimulate stem cells to relocate to sites of injury or damage?
V. What are the similarities and differences between embryonic and adult stem cells?
Human embryonic and adult stem cells each have advantages and disadvantages regarding potential use for cell-based regenerative therapies. Of course, adult and embryonic stem cells differ in the number and type of differentiated cells types they can become. Embryonic stem cells can become all cell types of the body because they are pluripotent. Adult stem cells are generally limited to differentiating into different cell types of their tissue of origin. However, some evidence suggests that adult stem cell plasticity may exist, increasing the number of cell types a given adult stem cell can become.
Large numbers of embryonic stem cells can be relatively easily grown in culture, while adult stem cells are rare in mature tissues and methods for expanding their numbers in cell culture have not yet been worked out. This is an important distinction, as large numbers of cells are needed for stem cell replacement therapies.
A potential advantage of using stem cells from an adult is that the patient's own cells could be expanded in culture and then reintroduced into the patient. The use of the patient's own adult stem cells would mean that the cells would not be rejected by the immune system. This represents a significant advantage as immune rejection is a difficult problem that can only be circumvented with immunosuppressive drugs.
Embryonic stem cells from a donor introduced into a patient could cause transplant rejection. However, whether the recipient would reject donor embryonic stem cells has not been determined in human experiments.
主題二: Stem Cell Markers

Human papillomaviruses (HPVs) are small DNA tumor viruses that cause benign and malignant tumors of squamous epithelia.(Fig1) Among all the subtypes of HPVs, HPV-16 and HPV-18 are identified as high risk HPVs which are mainly responsible for HPV correlated human cancer. HPVs contain a double stranded DNA genome of approximately 8,000 base pairs and up to 10 open reading frames (ORFs).(Fig2) ORFs within the early region code for proteins like E6 and E7 which are involved in the regulation of viral replication and the viral life cycle, HPV-16 E6 and E7 oncoproteins can abrogate negative growth regulatory signaling pathways of the host cell through interaction with p53 and pRB tumor suppressor proteins. As a result, high-risk HPVs infected cells proliferation become de-regulated, and then, transformation develops.
E6 oncoprotein
The E6 protein is one of the key cancer-causing proteins expressed by the Human papillomavirus (HPV). Among the strains of HPV known to cause physical changes associated with cancer and pre-cancerous lesions, three oncoproteins are recognized: E5, E6 and E7. Although low-risk HPV strains also produce these proteins, the four major high-risk strains—HPV-16, HPV-18, HPV-31, and HPV-45—all exhibit E6 and E7 proteins. E6's activity in the high-risk strains can be oncogenic, or cancer-promoting. Therefore, it is the strains which exhibit these proteins which are associated with cervical cancer and pre-cancerous lesion development in women. E6 is a 151 amino-acid peptide that incorporates a type 1 motif with a consensus sequence –(T/S)-(X)-(V/I)-COOH. It also has two zinc finger motifs.
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E6 is of particular interest because it appears to have multiple roles in the cell and to interact with many other proteins. E6 primarily causes cancer by associating with and thereby inactivating P53 or Rb proteins, which act as tumor suppressors. When tumor suppressor proteins are inactivated tumor growth proceeds unchecked. E6's interaction with p53 and Rb marks these proteins for degradation by ubiquitylation and ubiquitin ligase. E6 is proven to act on other cellular proteins, and to positively affect telomerase activity, thus inactivating one of the ways by which cells are normally prevented from dividing unchecked. Additionally, E6 can act as a transcriptional cofactor—specifically, a transcription activator—when interacting with the cellular transcription factor, E2F1/DP1. E6 can also bind to PDZ-domains, short sequences which are often found in signalling proteins. E6's structural motif allows for interaction with PDZ domains on DLG (discs large) and hDLG (Drosophila large) tumor suppressor genes. Binding at these locations causes transformation of the DLG protein and disruption of its suppressor function. E6 proteins also interact with the MAGUK (membrane-associated guanylate kinase family) proteins. These proteins, including MAGI-1, MAGI-2, and MAGI-3 are usually structural proteins, and can help with signaling. More significantly, they are believed to be involved with DLG's suppression activity. When E6 complexes with the PDZ domains on the MAGI proteins, it distorts their shape and thereby impedes their function. Overall, the E6 protein serves to impede normal protein activity in such a way as to allow a cell to grow and multiply at the increased rate characteristic of cancer. The full length HPV E6 genes encode a 160 amino acids protein, which contains two domains including zinc binding Cys-X-X-Cys motifs. High risk HPV E6 proteins both have anti-apoptotic activities and can interfere with the antiproliferative functions of p53, a tumor suppressor in cell proliferation. To do this, E6 first forms a complex with a cellular ubiquitin- protein ligase E6AP, the E6/E6AP complex then acts as a p53-specific ubiquitin-protein ligase to accelerate degradation of p53. Also, E6 can significantly reduce the half life of p53 in vivo from several hours to 20 minutes. Moreover, E6 oncoprotein interferes with the stabilization and activation of p53 in response to genotoxic and cytotoxic stresses. As a result, the negative growth regulatory function of p53 is effectively abrogated by E6-stimulated degradation in high-risk HPV-infected cells.
E7 oncoprotein
E7, a protein encoded by Human papillomavirus which inhibits pRb. Smaller than high risk HPV E6 protein, E7 proteins are about 100 amino acids in size and also contain 2 copies of Cys-X-X-Cys domains as well as E6. As an oncoprotein, E7 functions through forming complex with and promote proteolysis of hypophosphorylated pRB, the active form of retinoblastoma tumor suppressor gene product. Formation of E7/pRB complex interfere the complex formation of pRB with E2F. So E2F is released from inactivated combination with pRB and continues its work as a transcription activator to promote DNA synthesis and cell proliferation.(Fig. 3)
                                                     
Co-expression of high risk HPV E6/E7
In high risk HPV infected cells, E6 and E7 are usually co-expressed. The high-risk HPV E7 oncoproteins bind and degrade the pRB and the related pRB family members p107 and p130.17, as well as inactivate the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1.1820 As a result, there comes aberrantly increased expression of cyclin E, cyclin A and aberrant CDK2 activity mediated by E2F. E2F can also activate p14ARF to stabilze p53. As cooperation with E7 to promote cell proliferation, the high-risk HPV E6 oncoprotein induces the rapid proteasomal degradation of p53 by interacting with E6-AP, a host cell protein. By respective dysfunction of p53 and pRB tumor suppressor pathways, high-risk HPV E6 and E7 proteins immortalize and transform human cells effectively. (Fig. 4)
E6 / E7在cell line上的應用
<以下列文獻為例>
高危HPV所致宫颈癌的预防与治疗
(文章来源：丁香园)
子宫颈癌的发生与人乳头瘤病毒（HPV）感染密切相关。人体内确定的HPV有85种以上。其中与宫颈癌发生有关的有20多种，可分为高危型、中危型、低危型三种，其中高危型组的HPV16、HPV18等与宫颈癌有密切联系。现将高危HPV所致宫颈癌的预防与治疗总结如下：
1.制备基因疫苗
HPV基因功能区的L区包括L1和L2，L1晚期基因能刺激机体产生保护性抗体，因此该基因的克隆在制备基因工程疫苗方面成为可行性。Donnelly用编码乳头瘤病毒L壳蛋白的DNA疫苗，在兔体内的抗感染效果，说明了基因疫苗用于治疗也是一个有效途径。Conson J在防御16型乳头瘤病毒感染的疫苗中提到：子宫颈分泌物中存在的抗HPV16核壳蛋白的中和抗体可能具有保护作用，故而可制备HPV16疫苗以预防原发 HPV16感染。现在对这种疫苗的研究已取得相当进展，据澳大利亚布里斯班消息：美国PDA已批准在美国进行宫颈癌的人乳头瘤病毒Ⅱ预防疫苗的Ⅰ期临床试验。
2.构建HPV16型E6、E7基因反义质粒
E6、E7两个开放读码框架及其表达产物在宫颈癌细胞中常被检出，且这两个框架具有转化能力，E6、E7基因的表达与否是宫颈癌发生及维持的必要条件，故而研究E6、E7基因的反义质粒干扰E6、E7基因的表达成为一种治疗宫颈癌患者的手段。曾萍、司静懿等在人乳头瘤病毒16型E6、E7反义质粒对人宫颈癌细胞恶性的逆转作用中，采用构建可为地塞米松诱导表达的人乳头瘤病毒16型E6、E7反义质粒利用磷酸钙沉淀法将其分别转染到HPV16阳性的宫颈癌细胞Cask：和HPV阴性的宫颈癌细胞C-33A中，地塞米松诱导反义质粒表达后，Cask：细胞失去其恶性表型，而C-33A细胞生长特性及恶性行为未发生变化，证明了E6、E7基因反义质粒可成为一种治疗宫颈癌的方法。
3.重组病毒载体肿瘤疫苗的应用
由于HPV宿主细胞通过下调MHC1表达位点突变，下调抗原呈递，降低E6、E7抗原性，使肿瘤细胞免疫原性降低，逃避免疫系统的杀伤，这样使得肿瘤生物治疗效果很低，因此有人提出将肿瘤抗原肽的编码基因导入病毒载体，可能诱导出有效的杀肿瘤作用，故而有人把HPV16的E7基因重组于牛痘病毒表达载体，作为重组病毒载体治疗疫苗与E7蛋白分别免疫小鼠，均获得特异性CTL，据此 Boassneu MEG用表达HPV16E6、E7（经处理失去致癌性，保留抗原性）的重组病毒载体治疗免疫8例晚期宫颈癌病人，所有病人均产生抗牛痘LgG抗体，3人出现HPV特异性抗体，HPV特异性CTL在免疫疫苗后前两周可以检测到，有2例分别在15和21个月后仍保持临床良好。说明疫苗诱导的ADCC和CTL有一定效应，但仍存在E6、E7表达量较低，免疫原性不高的问题。但随着对重组病毒载体肿瘤疫苗研究的逐步深入使之应用于临床治疗将成为可能。
4.P53的应用
已经证实在人体内有抑癌基因P53，恶性肿瘤的发生均是致癌基因与抑癌基因相互作用的结果，HPV所致宫颈癌也不例外，关于P53与HPV的相互作用已在致癌机制中作了详细叙述。由于P53有很强的抑制细胞凋亡的能力，目前已被用于临床上作为基因治疗的一种方法。除此之外，P53蛋白的表达受DNA损伤的影响很大，而多数抗癌药物均属DNA损伤剂，因此P53的功能与肿瘤对某种特定抗癌药物的反应有很密切关系，P53在肿瘤中的突变会影响抗癌药物杀伤肿瘤的能力，有结果表明，含有突变型无活性的P53的肿瘤预后往往很差，由于P53与预后治疗上的重要关系，可以采用激活宫颈癌内已丧失活性的P53或重新引入有活性的P53，从而达到治疗宫颈癌的目的。
5.其它
目前较流行的治疗方式是局部有破坏的治疗，如刀切、激光灼烧、液氮冷冻、电热凝、三氯乙酸或者普达非伦脂腐蚀等。但这些方法只是针对可见的或病变的局部进行治疗，而HPV还可在临近接受治疗的组织中潜伏。因此，治疗后HPV感染的再发生率高。 5-Fu是限制HPV的DNA和RNA合成的细胞毒性药物，局部注射收效显著，但它可造成化学性阴道炎，长期使用还可导致阴道粘膜的损伤，甚至阴道狭窄。也有人利用干扰素的抗病毒、抗增殖和免疫调节等作用，通过肌肉和病变局部联合用药的方法治疗HPV收效比较显著，但关于HPV所致宫颈癌的预防与诊疗至今还没有一个十分完美和实际可行的方法，对HPV感染的预防以及如何迅速准确的诊断，安全有效的治疗有待进一步解决。