User:Kinkreet/SCRA/Methods

In vitro assays are always performed with clones from a single cell, this is to ensure results obtained will be true for the rest of clones in the stock. It is good for determining proliferation and differentiation potentials. The major downside of in vitro assays is that the identification of stem cells are always retrospective, and because the conditions are not physiological, the behaviour of the cells may differ significantly than in real life.

Organioids
Organioids are simply any structures grown in culture which resembles an organ, at least in terms of morphology and cell type composition. Organoids are often established from freshly isolated (using EDTA) glands from each organ. They can be used as models to study stem cells in each organ.

Identification of Stem cells
To characterize stem cells and progenitor cells, in vitro and in vivo assays can be performed. In vitro assays have the benefit of being highly-controlled, allows more detailed analysis of proliferation and differentiation potential; however it only allows retrospective identification and the behaviour of the cells may differ in vivo.

There are many types of stem cells, and they exists in small numbers, sometimes as sparse as 1 in 100,000. Their morphology is not distinguishable from its neighboring cells. So to identify stem cells, scientists must make use of 'markers' specific to each stem cell type. Markers are usually tested in combinations, and consists of different surface macromolecules, but can also be a cell's function, behavior, lipid content, transcription factors - basically anything that can distinguish one cell type from another. Using these markers, scientists can differentiate and sort stem cells out from the normal cell population; histochemical or fluorescence microscopy and fluorescence-activated cell sorting (FACS) are two main methods of identifying stem cells.

Below is a list of markers commonly used to identify different types of stem cells.
 * Lineage and stem cell markers - can be cell surface molecules/antigens or functions. e.g. MAC-2 (Macrophage marker); lipid content; transcription factors (NANOG - critically involved in the self-renewal of undifferentiated embryonic stem cell)
 * Lgr5 and Bmi1 are markers for intestinal stem cells.

Sphere formation assays
Non-adherant primary cells from certain tissues do not require an extracellular matrix to grow on, instead, they can form 3D spherical cultures in suspension. Stem cells and progenitor cells can give rise to spheres, but only stem cells are able to give rise to a series of spheres. Embryonic stem cells form embryoid bodies, neural stem cells form neurospheres, mammary gland stem cells form mammospheres, marrow mesenchymal stem cells from mesenspheres. Within each sphere, there will be a range of cells with varying levels of differentiation. Sphere forming assays are good for qualitative determination of the presence of stem cells in a poorly-characterized tissue.

Clonal analysis
One can induce the differentiation of some stem/progenitor cell to determine the cell's potency, as well as determining what factors are required for the stem cell to commit to a specific lineage. For example, hematopoietic cytokines can instruct lineage choice. The lineage can be determined by using cell type-specific markers as listed above.

Clonal analysis of tumours can also be performed to find mutations which causes the tumour to be more aggressive. Single tumour cells is plated and allowed to grow into a colony, this is then subjected to carcinogenic environment which encourages mutations; any mutations which causes a cell to be more aggressive will cause that cell to proliferate more, forming a bulge. Cells from the bulge can be isolated and the mutation identified, possibly identifying the gene which is involved in tumorigenesis, or the prevention of it.

Microscopy
The animal or organism is fixed and labelled with a tagged antibody. The labelled slide is then viewed under the microscope to identify the stem cells. The advantage of identifying stem cells in this way is that we can see where the stem cells are in relation to the rest of the tissue. The disadvantage is that the cells cannot be reused or studied as they are killed.

Functionl assay
The growth potential of a single cell can be estimated by counting the number of colonies formed from plating a single cell (technique known as colony forming assays, or clonogenicity assays). Holoclones have the greatest reproductive capacity (less than 5% of the colonies terminally differentiate); paraclones have a short replicative lifespan (>15 generations before terminally differentiating); meroclone contains a mixture of holoclones and paraclones, and is the transition state between the holoclones and paraclones.

As stem cells have very high replicative potential, when plated they able to proliferate, giving rise to many clones (holoclones); because they can also self-renew, the holoclones themselves can be replated indefinitely to give rise to more holoclones. Transit amplifying cells are usually meroclones or paraclones. This the cell type can be identified by the type of clones the plating produces. Unsurprisingly, the proportion of holoclone cells generally decreases with age.

Lineage tracing can also be used. A single progenitor- or stem cell labelled using lineage tracing will give rise many progeny, which will also be labelled. However, only patches arising from stem cells will persists as they have virtually unlimited replicative potential.

Fluorescence-activated cell sorting (FACS)
Fluorescence-activated cell sorting is a technique introduced in 1972 by a group from Stanford University. Cells in tissues or on culture flasks are separated and treated with a fluorescently-tagged antibody against the specific stem cell marker. These antibodies will bind to the stem cells only, as only they expresses the marker. The cell suspension is then diluted and forced through a very narrow nozzle; the nozzle is narrow so that only one cell can pass through at a time. After exiting the nozzle, the cell is tested for fluorescence with the help of a laser. Any cells which are fluorescent (a stem cell) will generate an electric signal due to its fluorescence, and is deflected in an electric field into a separate tube from the rest. Every cell in this tube will contain fluorescent cells and are thus stem cells.

The advantage of identifying stem cells in this way is that identification is categoric and not subjective, and that the identified cells can be used in further experiments, as they are still alive. The disadvantage of this is that a marker must be chosen that is expressed on the cell surface, and it is not possible to see the localization of the stem cell in relation to the rest of the tissue.

RT-qPCR
There are genes and transcription factors that are differentially-expressed in stem cells. Real-time Quantitative PCR can be used to analyse the level of expression of these genes in stem cells relatively to other cells. Roughly the same number of cells are lysed and the RNA (containing the mRNA) extracted, a reverse transcriptase is used to synthesis cDNA from RNA. Specifically-designed primers can then be used to amplify the gene of interest (GOI) which should reside within the cDNA. The higher the level of expression of the gene, the higher the levels of mRNA and thus higher levels of reverse-transcribed cDNA, and thus more PCR products. The amount of PCR products from stem cell lysates compared to normal cell lysates will give us an indication of whether the GOI is differentially expressed in stem cells.

Genetic engineering
Using recombination technology, a reporter gene, such as GFP, can be inserted after a stem cell-specific promoter, or be tagged to a stem cell-specific marker. Thus, undifferentiated cells will express the marker, while differentiated cells will not. Thus, combined with real-time microscopy, it is possible to track the movement of stem cells as the organism develops. Later, this method can be followed by FACS to isolate the stem cells for further studies.

Transplantation assay
Gives an indication of the proliferative and potency of cells (a single stem haematopoietic stem cell is able to reconstitute the entire bone marrow, and the reconstituted population of stem cells are able to reconstitute further bone marrows). Adult stem cells will also give rise to a limited of lineages, and these are apparent in a transplantation assay (epidermal stem cells will only give rise to hair). However, transplantation assays are time-consuming and laborious.

Primary cell culture
Primary cells are cells straight from the tissues of an animal. The tissues are dissociated using mechanical, chemical and physical methods, to obtain a single cell suspension which is then grown in a media containing a specified cocktail of cytokines, growth factors, hormones, serum and feeder cells.
 * Bone marrow can be obtained in smaller bones by crushing the bones and pass it through a filter which capture all the bone fragments; if the bone is bigger, the bone marrow can be drained.
 * Human cord blood cells - cord blood is present in the placenta and umbilical cord and can be collected from the umbilical cord at the time of birth and stored; cord blood contains a high levels of stem cells which can be used for future treatments of the child. Ficoll-Paque is used to separate blood into its components. Ficoll-Paque is added at the bottom of a conical tube, and the blood is slowly layered on top. The tube is centrifugated, and the blood components will separate into fractions in the order, from top to bottom: plasma and other constituents, a layer of mono-nuclear cells called buffy coat (PBMC/MNC), Ficoll-Paque, and erythrocytes & granulocytes which should be present in pellet form.
 * Human and mouse epidermal cells - obtained by separating the epidermis from the dermis using EDTA, mincing it and then separating the cells using trypsin-EDTA (to degrade extracellular proteins used for adhesion, as well as chelating calcium, which is required for integrin- and cadherin-dependent cell-matrix and cell-cell adhesion, respectively)
 * Tumour cells can be dissociated by mincing, treating with EDTA and collagenase.
 * Feeder cells are adherent, growth-arrested, incapacitated (by irradiation or chemicals to stop their proliferation) cells whose purpose is to provide a substratum for other cells to grow onto, and provides an intact extracellular matrix, cytokines and other factors. Most feeder cells used are mouse embryonic fibroblasts (MEF). Even though it is widely used, feeder-free cultures are preferred when possible as the exact cocktail of nutrients and signals produced by the feeder cells are unknown, and thus add variability to the experiment. Furthermore, the feeder cells are themselves animal cells, which can harbor pathogens.
 * Embryonic stem cells are easy to culture, and in vitro tend to differentiate into different types of tissues. Adult stem cells are hard to culture. Most cells can be grown on fibronectin-coated or Matrigel-coated plates, but some requires a complete extracellular matrix provided by the feeder cell.

FACS
Nuclear labels - Cells can have nuclear stains (e.g. DAPI, PI, 7AAD/Hoechst) applied to them and ran through FACS; cells which are replicating will likely to have two sets of the genome and thus will give a brighter signal in the FACS machine. Cells which only has one set of DNA can be sorted out and should contains stem cells, albeit in a huge cocktail of other cells. This is a crude and non-precise method of isolating stem cells, and should be used as a first stem of many.

Methods for studying proliferation
Terminally differentiated cells (post-mitotic) do not proliferate and stem cells proliferate at low levels, the levels of proliferation is highest in progenitor cells. During stem cell proliferation, the resulting progeny from a stem cell population must be around 50% stem cells and 50% progenitor cells; this is to ensure the self-renewal property of stem cells. At the single cell level, both symmetric and asymmetric divisions are observed. Divisions based on environmental factors can give rise to symmetric or asymmetric divisions, it all depends on the local microenvironment which determines the fate of the cell, such as when the progeny escape the stem cell niche, it will differentiate, otherwise it will stay a stem cell. Sometimes the microenvironment a cell finds itself in depends on the orientation of the mitotic spindle, which determines the axis in which chromosome are separated and ultimately the position of the two daughter cells. Divisions based on a preset program divides asymmetrically regardless of the environment. The programmed hypothesis falls short when thinking about damage and repair, when there is a wound, the stem cell divides to give progeny with a higher proportion of differentiated cell to replace the old ones.

In reality, it is both environmental and programmed. The fate of the cells is largely dependent on the microenvironment, but the orientation of division is not random. For example, cells in the basal layer of the epidermis (including stem cells) divides towards the outside, with cells in the intermediate layer mostly dividing laterally.

BrdU labelling
Bromodeoxyuridine (BrdU) is an analogue of thymidine; if it is present, it will be randomly incorporated into the genome in place of some thymidines. Thus cells which replicate more will incorporate more BrdU, which can be detected ??. Stem cells, although they do not replicate much, can incorporate BrdU into the DNA through a pulse of BrdU. When BrdU is removed, every successive division of the cell leads to the dilution of BrdU in the progeny. Thus, assuming that initially all cells have the same level of BrdU, the concentration of BrdU in a cell is inversely proportional to the number of times it replicated, which is an indication of the level of proliferation of that cell.

Cyclin immunostaining
Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein and promotes progression through the G1-S phase of the cell cycle ; similarly, cyclin E-Cdk2 activity is highest during G-S transition. Thus, we can immunostain of these cyclins to identify cell populations which are active in the cell cycle, and these populations are not likely to be stem cells, which are quiescent. This method is not particularly stringent as proliferating cells not transitioning will not be stained and can be mistaken for a stem cell; furthermore, the population of stem cells are likely to be small, and so not easily identifiable.

Ki67 staining
Cyclin immunostaining stains for cells only while they are transitioning between different phases. Ki67 is an antigen expressed in all activate stages of the cell cycle, and so staining for Ki67 will identify cells which are replicating and not likely to be stem cells. The major disadvantage of this technique is that we still do not know what Ki67 is.

Stem cells are defined as cells which can self-renew indefinitely and give rise to differentiated cells, and so a stem cell's replicative potential is very high; however, in vivo, stem cells do not replicate often, and most of the time exists in quiescence. The question then is "What makes stem cells enter the cell cycle from G0?"

Label retaining cells (LRC) assay
This is essentially a pulse and chase experiment, whereby a short term administration of a label is applied to highlight dividing cells. After his the label is removed; in subsequent divisions, the concentration of the label is diluted between the progeny. Thus the more proliferative a cell is, the more the label is diluted. Those cells which retain the label do not divide very much, which is a characteristic of a stem cell.

However, a principle flaw with this argument is that the label can only enter dividing cells; if stem cells are not dividing, then the label would not be incorporated in it in the first place. One solution is to label the cells early on in development while the stem cells are still proliferating. However, any cells which retain the dye is not proliferative, and it might have just been that at the time of the pulse, it was dividing.

This label can be BrdU, but BrdU is toxic and detection requires fixation and permeabilisation; furthermore, it is difficult to detect intermediate levels of proliferation. An alternative to Brd LRC assays is to use Histone 2B (H2B) fused with GFP. H2B is part of chromatin and have little toxicity. The H2B-GFP construct can be introduced under the regulation of a tetracycline-inducible promoter, and is induced in significant amount over the endogenous population. The H2B-GFP is incorporated in the pulse into the chromatin, and is diluted as the cell give rise to progeny. Having the GFP tag also means FACS can be performed on live cells, which can give rise to relatively pure batch of cells to be studied further at a later stage. Doxycycline (a member of the tetracycline antibiotics group) promoter are also used.

Direct Observation
Lineage tracing is the identiﬁcation of all progeny of a single cell. This occurs by labelling a cell with a single marker which will persists into future generations, will not be transferred to other cells that is not its progeny, and persists without being diluted. Historically, the cell lineage of cells were painstakingly determined by time lapse microscopy; the cell fate of every cell of the nematode Caenorhabditis elegans was determined this way. The advantage of direct observation is the ease of the technique and the assurance that no chemical and physical factors (apart from light) is interfering with the cells. A major limitation is that transparency of the tissue is required for observation of cells not on the periphery,; this is why many studies are performed in zebrafish embryo, which is transparent.

Cell Labelling
When direct observation is not possible, the next step is to label the cells of interest. Vogt used vital dyes (dyes which do not kill the cells) to label and trace cells in amphibian embryos. More recently, carbocyanine dyes such as octadecyl (C18) indocarbocyanines (DiI) and oxacarbocyanine (DiO) are used to mark the plasma membrane and the progeny is followed this way. The lineage tracing of the neural crest of chicken embryos and the frog Xenopus laevis were done using carbocyanine dyes. Fluorescein-conjugated dextran and horseradish peroxidase (HRP) can be injected directly into embryos (provided it is large enough, such as in Xenopus) or at a later stage, to trace the progeny by following these agents. Dextran and HRP are markers which are too large to pass through the gap junctions and so cannot pass between cells, only passed on, and diluted, through its progeny.

Genetic Markers
Direct observation and labelling using dyes are now largely obsolete, and is partially replaced by introducing genetic markers into cells through electroporation, injection, transfection and/or viral transduction. The advantage of using genetic markers is that the reporter is passed on to the progeny without dilution at the protein levels. Limitations of genetic markers is the relatively low efficiency of incorporating the introduced gene into the genome; this can be overcome by also introducing, in parallel, a selectable marker to select out only those cells which transfection or transduction worked; viral vectors can also be used in place of plasmids to increase efficiency. Furthermore, introducing foreign genetic materials will alter the cell behaviour, whether this is significant or not is unknown.

One must beware that some tissues are known for spontaneous cell fusion, and so markers, even genetic markers, can be passed on to cells not part of the cell lineage, and can lead to the illusion of transdifferentiation (the change of cell type from one lineage to another without first undergoing an intermediate pluripotentstate or progenitor cell type.

Transplantation
Transplantation is another strategy for lineage tracing. Cells are transplanted into the host and its progeny is traced by observing physiological differences (such as cell size) between the host cells and the transplanted cells. If the host cells and the transplanted cells do not have any obvious physiological differences, the cells to be transplanted can be incubated in tritiated thymidine, which is radioactive, and then transplanted into unlabelled host embryo. However, radiation is involved which can damage the embryo; furthermore, the exact cells cannot be determined accurately. The limitation of transplantation for lineage tracing is the requirement for surgery, the creation of a wound, and that the cell may not behave in the graft as it would normally in tissue.

Genentic recombination
Lineage tracing by genetic recombination is now the preferred choice. It involves expressing an recombinase which is under the regulation of a cell- or tissue-specific promoter; the recombinase then activates a reporter gene. The advantage of this system is that marking is permanent and cell type or tissue-specific. Cre-loxP from bacteriophage P1 and FLP-FRT from Saccharomyces cerevisiae are the two most widely employed recombinase systems. The FLP-FRT system acts interchromosomally and is mainly used in Drosophila while theCre-loxP system is intrachromosomal and is mainly used in mice.

In the FLP-FRT system, the flippase enzyme is expressed under a heat shock-inducible tissue- or cell-speciﬁc promoter. Flippase acts to recombine an ubiquitous α-tubulin promoter with a reporter gene on the homologous chromosome, at the ﬂippase recognition target (FRT). In the Cre-loxP system, a mouse line with the Cre recombinase under the regulation of a tissue- or cell-speciﬁc promoter, is crossed with another mouse line with the reporter gene downstream of a loxP-STOP-loxP (‘‘ﬂoxed’’ STOP) sequence, both of which are under the regulation of an ubiquitous promoter (usually the Rosa26 locus). In most tissues of the crossed mice, the "floxed" STOP sequence contains a STOP codon which prevents the reporter gene from being transcribed, in those tissues corresponding to the tissue- or cell-specific promoter, the Cre recombinase is expressed, and Cre acts to excising out everything between loxP, excising out the STOP codon, which permits the reporter gene to be transcribed. The tissues in which the reporter is expressed depends on the promoter used; for example, Sox9 is a promoter specific to follicle bulge stem cell. The ubiquitous promoterRosa26 can be improved by adding an exogenous strong promoter CAG; furthermore, it can contain a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) which stabilizes the mRNA.

The expression of the Cre recombinase can be induced spatially and temporally. The Cre recombinase can be fused with the human estrogen receptor (ER) to form CreER. In the absence of ER ligands (estrogen 17β-oestradiol, tamoxifen, 4-hydroxy-tamoxifen (4-OHT)), CreER is kept in the cytoplasm by binding to heat shock proteins (hsp). In the presence of ER ligands, the ligands diffuse into the cytoplasm and binds to CreER, which causes a conformational change leading to release from hsp. This allows CreER to translocate into the nucleus where it can recombine the loxP sites. However, endogenous ER ligands exists, and so mutants of CreER has been made to prevent expression due to these low levels of endogenous ligands; for example, the most widely used version of inducible Cre is CreERT2, in which Cre is fused to a mutated ligand binding domain of the human estradiol receptor (ERT2), which does not bind endogenous estradiol but is highly sensitive to nanomolar concentrations of tamoxifen or its metabolite 4-hydroxy-tamoxifen (4OHT). A similar method of Cre induction is to fuse it to progesterone receptor to make CrePR. PR ligands anti-progestins Org 31376 or Org 31806 are used to induce the translocation of CrePR into the nucleus. A mutated version can also be activated by RU486 (mifepristone). Similarly, mutants have been generated to prevent activation from endogenous progesterone. However, leakiness is still a common problem and so control experiments must be carried to determine the levels of non-specific activation. Another problem is that recombination is not stringent, and can happen during development or in the adult. These two problems can be overcome by using a mutant where two signals are required; for example, the AhcreERT transgenic line is under the control of the Ah promoter (activated by β-napthoflavone) but also requires Tamoxifen binding.

Sox2 is a stem cell marker, and the Sox2 promoter can be used as a stem-cell-specific promoter to express Cre. The activation of Sox2+ cells are not high, in mice only about 20% of tamoxifen (TAM)-treated cells expresses the reporter gene ; thus to permanently label Sox2-expressing cells and their progeny, often consecutive days of TAM treatment is required. This can apply to other promoters.

Multicolour Genetic mosaic
Genetic mosaics are where somatic cells with different genotypes resides in the same animal. By knocking out genes of interests differentially (both spatially and temporally), one can study the effect of the gene in specific tissues/cells during specific time.

The "Brainbow" mouse contains many random fluorescent genes, each flanked by incompatible loxP; Cre recombinase is expressed in tissue- or cell type-specific cells, but, by chance, excise and recombine at different combinations of loxP pairs, meaning some cells will contain more genes of one colour than other cells, and vice versa. This gives cells a multitude of colours depending on the loxP pairs.

Mosaic analysis with double marker (MADM)

Lineage tracing is able to inform us of the location, number and differentiation status of a cell's progeny. It has advantages over other techniques as one is able to study cell behaviour in intact organisms or tissues, especially if the organism is transparent in colour, such as some species of zebrafish. In the future, it is likely that lineage tracing will use activation of signalling pathways as a trigger.

Transcriptomics
The transcriptome is a complete set of transcripts (including mRNAs, non-coding RNAs and small RNAs) in a cell, and their quantity, for a specfic developmental stage of physiological condition. Transcriptomics also study the splicing patterns and other post-transcriptional (but pre-translational) modifications made to the transcript.

Microarrays
One way to see the differential gene expression is to use gene expression profiling. Gene expression profiling uses DNA microarray to measure the level of expression of thousands of genes at once. A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (orreporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.

A drawback of this technique is that a large amount of RNA is required to produce enough cDNA to hybridize with the microarray. A partial answer to this is to use PCR to amplify the cDNA before hybridizing, however this can induce mistakes and blur out the quantitative measurements; also, you need to have a gene of interest to perform PCR in order to design the primers, as using random primers is not desirable.

RNA-Seq
RNA-Seq is a transcriptome profiling technique which utilizes deep-sequencing technology. RNA-Seq allows more precise measurement of the levels of transcripts, including their isoforms, than other techniques such as hybridization- or sequence-based approaches. In the hybridization-based approach, RNA is isolated from the cell lysate and reverse transcribed using fluorescently tagged deoxynucleotides to give a library of fluorescently-labelled cDNA representing the RNA content of the cells. The fluorescent cDNA is then incubated on a DNA microarray. If there is a cDNA sequence which is complementary to the probe on the microarray, it will hybridize. The more hybridization that occurs, the bigger the fluorescent signal, and this means the levels of the corresponding transcript in these cells is higher than the rest. The microarray can be custom made to include only the genes (and their isoforms) of interest; it can also be probes spanning exon junctions to look at different splice variants. Hybridization-based techniques are high-throughput and relatively inexpensive, but requires prior knowledge about the genome sequence and high background from cross-hybridization, and inaccurate quantification owing to saturation of the probes or the insensitivity to low signals. Thus, RNA-Seq is appropriate in determining levels of transcript even when the RNA levels in cells are low, especially in stem cells which have a small nucleus.

In sequence-based approaches, the cDNAs are sequenced. The Sanger sequencing of cDNA or EST librarys are low throughput, expensive and not very quantitative; advances from this such as serial analysis of gene expression (SAGE), cap analysis of gene expression (CAGE) and massively parallel signiture sequencing (MPSS) are tag-based methods which provides high-throughput and precise gene expression levels. However, tag-based techniques are expensive and isoforms cannot be distinguished from each other because only a portion of the transcript is analysed.

Overview
Long RNAs are converted to short cDNA fragments either by RNA fragmentation (RNA hydrolysis or nebulization) followed by reverse transcription, or by reverse transcription then cDNA fragmentation by DNase I treatment or sonication (small RNAs such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and short interacting RNA (siRNA) do not require this fragmentation step). Sequencing adaptors are attached to the resulting cDNA fragments to one or both ends. The cDNA is then sequenced from one (single-end sequencing) or both ends (pair-end sequencing); each run usually lasts for 30-400 bp. The sequences obtained is aligned with a reference genome or transcripts, or assembled de novo (like a jigsaw puzzle without the finished image). They are then classified as either exonic reads, junction reads and poly(A) end-reads. The relative number of a particular sequence represents its level of expression, and the relative number of times a base pair is sequenced within the transcript gives base-resolution expression profile for each gene.

RNA-Seq have advantages over hybridization-based approaches as it does not require the transcript to be part of any existing genomes, and so no prior knowledge is required. Because RNA-Seq uses sequencing technologies, even single nucleotide polymorphisms can be detected, whereas it will not be detected in hybridization-based approaches, as complementarity does not have to be absolute. RNA-Seq have no background and there is no upper limit to the levels that can be quantified, unlike hybridization approaches which is limited by the saturation of the probes. The accuracy of RNA-Seq has been determined to be accurate using qPCR. RNA-Seq is also highly reproducible.

Transplantation
Cells (or single cell) can be transplanted into NOD/SCID mice and observe for tumour formation. It has been found that the more immunocompromised the mice (e.g. in Il2rg KO) is, the faster the graft; thus, the number of tumour cells predicted within a tissue depends on the protocol used, where some are more efficient than others.

These cells can be diluted more and and more in injected into the NOD/SCID mice to correlate the frequency of tumour formation. The relationship follows a Poisson linear regression, where the higher the dose, the higher the number of mice developing tumours, but the progressively higher doses have progressively less effect.

Suicide ablation and rescue
The concept behind this technique is to induce killing of the stem cells, and then remove the killing to allow the remaining stem cells to recover and reconstitute the damaged tissue.

This technique involves making a transgenic which encodes a suicide gene which expresses a protein that results in killing of the cell, when induced by a compound. The suicide gene is usually an enzyme which converts a non-toxic pro-drug into highly toxic metabolites. Thus cell killing can be induced by adminstering the pro-drug, and any cell which expresses the enzyme will be killed. Thus, if the suicide gene is under the regulation of a cell type-specific promoter, such as Sox2 for stem cells, then all Sox2+ cells will be killed.

The most common combination of suicide ablation uses the Herpes simplex virus thymidine kinase (HSVtk) gene as the suicide gene, and ganciclovir as the pro-drug. Ganciclovir is a synthetic analog of 2'-deoxy-guanosine, and can be phosphorylated by the gene product of HSVtk to be come an analog of dGTP, this is incorporated into DNA when cells replicate, this inhibits DNA synthesis and leads to cell death. The advantage of this system is that it has shown to be safe and effective in abrogating all graft-verse-host disease. The disadvantage is that more proliferative cells are more likely to be killed, as it requires the phosphorylated GCV to incorporate into the DNA, and so killing of the cell is not stringent. Other suicide gene/pro-drug systems exists, including CD20/anti-CD20mAb, [FKBP-FAS/AP20187, AP1903 dimerizers] and FKBP-caspase9/AP20187 dimerizer pro-drug.

The pro-drug can be administered through an osmotic pump. For example, GCV administration uses 2μM GCV dissolved in 0.9% saline, and released 2mg a day.

As the ablation is not stringent or complete, there will be some cells that are not ablated. If they are stem cells, after the removal of the pro-drug (surgically. Marks the start of the rescue) will lead to the long-term reconstitution of the tissues from these cells.