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No, integration of the microinjected transgenic DNA into the embryonic genome is not very efficient. Therefore, only about 10-30% of pups will have inherited the transgene. Since integration most often occurs prior to the first zygotic cell division, the transgene will be present in all nucleated cells of those mice, the founder (F0) mice, that inherit the transgene. While all of the founder mice carry the exact same transgene, integration of the transgene is a random event. Therefore, each founder is considered unique with respect to the chromosomal location or integration site of the transgene, the number of transgene copies integrated at that site, and the expression pattern of the transgene.
No, the chromosomal environment at the transgene integration site can significantly influence transgene expression, a phenomenon called position effect variegation. For example, if the transgenic DNA integrates into heterochromatin, which is transcriptionally inactive, the transgene will not be expressed in the adult mouse. As integration is a random event, transgenic expression levels and patterns can vary substantially among founders.
Sequences within the transgene can also effect expression. Vector sequences of 100bp or more may be recognized as foreign and target the locus for transcriptional inactivation, whereas the inclusion of heterologous introns or insulator sequences can enhance expression.
Occasionally, the DNA integrates after the first cellular division resulting in some tissues/cells that carry the gene and others that do not. These mosaic founder animals may transmit the transgene through the germline at very low frequencies.
Remember that each founder, although carrying the same transgene, is unique with respect to copy number and integration site. For these reasons, unless there is a specific purpose, breeding one founder to another is not recommended.
The relative levels and tissue-specific pattern of transgene expression must be established for each founder line. Expression patterns are commonly analyzed in F1 mice as transgene expression products, such as mRNA or protein, and often require the isolation of tissue. This information will help to determine which founder lines should continue to be bred for proposed experiments. For instance, tissue-specific expression may not be tightly regulated in some founder lines resulting in non-specific or mis-expression of the transgene in various tissues. Expression levels may be too high or too low in other founder lines making these lines unsuitable for further analysis.
Founder mice can, in general, be bred to any desired strain. We recommend mating wild-type mice, not non-transgenic littermates to founders initially to begin establishing the line and generating F1 for expression analysis. Mixed B6:SJL founders can be kept on a B6SJLF1 background or crossed with C57BL/6 mice if interested in backcrossing the transgene onto a pure background strain.
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ES cells are grown in selection media for 6-10 days following electroporation. Approximately 200-250 clones that survive selection, and have therefore integrated the targeting vector DNA, will be provided in a 96-well plate format. You will need to isolate DNA from each well and assess the DNA to determine whether the targeting vector integrated randomly or via homologous recombination.
Following drug selection, colonies are picked into a 96 well plate and allowed to grow for several days. When confluent, the cells in each 96 well plate are split into 4 replica 96 well plates. Two plates are frozen and stored at –80°C for future use. The other two are placed back into the incubator and cells continue to grow until wells are slightly overgrown. Wells are then washed with PBS, frozen, and provided for DNA isolation and analysis.
We recommend isolating DNA from one replica plate initially. Expect to isolate enough DNA from each well for a PCR reaction and one restriction digest. If precipitated DNA is resuspended in 40µl/well of buffer, 1µl is used for PCR and 30µl for a Southern leaving a bit of DNA to spare. All wells can be screened initially by PCR for homologous recombination at one arm. PCR positive clones are then screened by Southern for recombination at the remaining arm. The second 96-well plate is used for a second Southern (if Southerns are used to analyze both ends) or as a back-up.
ES cell clones initially frozen in the 96 well plates are stored at –80°C during the screening period. Cells kept under these conditions are not stable over long periods of time and should optimally be thawed within 2 months. This is one of the reasons it is so important to have a well-defined, robust screening assay prior to electroporation.
Potentially targeted clones identified in the initial screen are thawed, expanded, frozen and stored under stable conditions in LN2 dewars. It is important that these clones are confirmed by Southern as correctly targeted at both the 5' and 3' ends prior to microinjection. This ensures that the correct clones have been thawed and are of interest. It would be unfortunate to discover 6 months down the road that the mice you have been working with do not have the expected mutation. Additional DNA from the thawed/expanded clone(s) will be provided at this step.
Creation of germline competent chimeric animals involves the microinjection of pluripotent embryonic stem (ES) cells into the cavity of an expanded 3.5 d.p.c. blastocyst stage embryo. ES cell lines are initially derived in vitro from the outgrowth of the inner cell mass (ICM) of the blastocyst, the portion of the blastocyst that gives rise to the embryo. Thus, when injected back into a blastocyst, the ES cells have the ability to incorporate into the ICM and contribute to the genetic makeup of the developing embryo. The resulting pups are considered chimeric in their genetic makeup as they consist of tissues deriving from both the microinjected ES cell and the endogenous ICM of the host blastocyst genome. The desired outcome of this process is to create chimeric mice that inherit germ cells derived from the microinjected ES cells. Using this technique, mutations can be introduced into the ES cells in vitro, then incorporated in vivo into the germline of a mouse and transmitted from generation to generation.
There is no way to predict a priori the pluripotent potential of a given ES clone prior to microinjection. While clones that look beautiful in culture exhibiting characteristics of undifferentiated cells is promising, visual appearance alone is not indicative of pluripotency. Individual ES cell clones vary in their ability to contribute to the genetic makeup of a chimeric mouse. Good clones are capable of consistently generating highly chimeric males while other clones may generate only low percentage chimeras or none at all.
A chimera is defined by Wikipedia as an animal that has two or more different populations of genetically distinct cells that originated in different zygotes; if the different cells emerged from the same zygote, it is called a mosaic (which happens occasionally in transgenic founders but not in ES cell derived chimeras).
ES cell chimeras are comprised of cell populations and tissues arising from both the host blastocyst strain and the ES cell clone introduced into the E3.5 blastocyst stage embryo. While ES cell contribution to organs and tissues is not immediately evident and must be determined experimentally, ES cell contribution to pigmented tissues such as eyes, skin and hair can be visualized in neonatal pups. An indication of ES cell contribution to coat color can be visualized at about postnatal day 10 when the hair begins to come in. For HM1 (129P2/Ola) derived pups, ES cell contribution is notable as early as postnatal day 3 as marbled color skin patterns emerge. For some C57BL/6 ES cell derived pups, chimerism is visual at birth by eye color.
The percent chimerism of each mouse is determined at weaning and refers to the degree of coat color deriving from the ES genome. The greater the ES cell contribution, the higher the percent chimerism. For example, a 95% 129-derived chimeric male will have only about 5% pure black color inherited from the host blastocyst cells and the rest will be non-black.
Coat color chimerism indicates that individual ES cells were incorporated into the ICM of the blastocyst following microinjection and that, once incorporated, were capable of giving rise to various cells or tissues in the resulting animal.
As both germ cells and melanocytes (pigment producing cells of the skin, hair and eyes) arise from migratory precursor cell populations within the developing embryo, the percent of 129 ES cell derived coloration in the coat is often correlative with the inheritance of ES cell-derived germ cells. For example, 129P2/Ola derived chimeras have marbled coats (and skin) of chinchilla, white and agouti (Mouse Strain Coat Colors). Thus, the higher percentage of ES cell derived coat coloration, or chimerism, the greater the likelihood that the incorporated ES cells were also able to generate germ cells that migrated to and populated the germinal ridges.
Coat color chimerism and germline transmission is less correlative with C57BL/6 ES cell lines.
No. Each chimeric mouse, even those created from the same ES cell clone and regardless of percent chimerism, is unique in its ability to transmit ES cell inherited genes. Germline transmission of ES cell derived genes in 129 derived chimeras mated to wild-type C57BL/6 is indicated by the production of agouti pups. It is not unusual for highly chimeric males (90-99% chimerism) to be sterile, or to produce many black pups before generating agouti pups. Some fertile males will never produce any agouti pups. In our experience, highly chimeric HM1-derived males often, but not always, generate agouti pups in the first or second generation. Agouti coat color only indicates the ability of the ES cell to give rise to germ cells and agouti pups must be genotyped for the inheritance of the mutated allele.
Chimeric mice can be mated with virtually any strain desired. However, we recommend mating highly chimeric males to wild-type females of the blastocyst strain initially to assess the frequency with which ES cell derived genes are transmitted by a particular chimera. This is particularly true for conditional KO lines. Coat or eye color of offspring indicate germline transmission when blastocyst strain animals are mated with 129-derived and B6-derived chimeras, respectively. When using these strain combinations, only the pups with ES cell derived coloring need to be genotyped.
If using other mating schemes or strains in which color cannot be used as a indicator of ES cell germline transmission, all pups must be genotyped.
No, much simpler and faster PCR-based genotyping strategies should be used at this point. The elaborate screening assays developed to detect 5' and 3' targeting events in the ES cell clones are not necessary at this stage, as homologous recombination in a particular ES cell clone should have been confirmed prior to microinjection.
When starting, keep in mind that each chimeric male, even those created from the same ES cell clone, is unique in its ability to transmit ES cell inherited genes. It is not unusual for chimeric males to be sterile or to produce many black pups before generating agouti pups. Some fertile males will never produce any agouti pups. Therefore, it is advantageous to begin breeding several of your best males initially. It is also wise to breed males generated from at least two different clones if available.
In general, highly chimeric females should be bred only if male chimeras are not available. Females occasionally produce agouti pups but the frequency is usually very low. However, if females are the only chimeras available—by all means, give it a go.