Additional Gene Targeting Services
Gene Targeting Tail Biopsy Service
- Chimeric offspring will have tail biopsies between 10-21 days of age. Approximately 0.5 cm of tail tissue will be removed from each mouse and placed in a numbered tube that corresponds to the number of the mouse.
- These samples will be frozen and shipped, at the client's expense, on dry ice, by overnight shipping service.
- The client is responsible for purifying the DNA from the tail sample (see DNA purification protocol), performing the genotype analysis and reporting the results to the VARI Germline Modification Core within two weeks of receipt of the DNA.
- All mice will be shipped to the client when the mice are 6 weeks of age whether or not the genotyping reactions have been completed, at the client's expense.
- Clients requesting germline testing of the chimeras are required to submit a separate request for this service.
- Clients experiencing problems with the genotyping procedure are encouraged to contact the VARI Germline Modification Core for assistance and advice.
Tail DNA Isolation (AutoGenprep 960)
The AutoGenprep 960 is a fully automated high throughput instrument for DNA extraction in the 96 well format, employing the proven solution phase organic extraction method. The 960 combines an automated 12 channel pipetting module, robotic plate movement and a built-in centrifuge to provide high throughput extraction.
Preparation of Mouse Embryonic Fibroblasts (MEF's)
Primary embryonic fibroblasts, isolated from E13.5 day embryos, can be used for a variety of applications, including elucidation of molecular mechanisms and analyses of growth, senescence, apoptosis, and differentiation. Also, if a gene knockout results in an embryonic lethal phenotype, analysis of the gene function can still be undertaken at the cell biological or molecular level using the fibroblasts from individual mutant embryos.
Preparation of ES cell lines from blastocysts
Individual mutant blastocysts can be grown in culture and passaged to generate stem cell lines. This technique is useful in gene knockout experiments where early embryonic lethality occurs. The 3.5-day embryos can be isolated and the resulting stem cell lines can then be characterized and subjected to further analysis.
PCR analysis of blastocysts
In some instances, liveborn heterozygous or homozygous knockout pups are impossible to obtain. For this situation, DNA can be isolated from individual blastocysts and PCR analysis can be performed to determine the genotype.
Cre-Lox Procedure
Conventional germline knockouts sometimes result in embryonic lethality or may not necessarily represent the best approach for studying the function of a particular gene in vivo. Conditional knockouts result in the lack a particular gene only in a specific tissue or during a specific stage of development. This can be achieved by electroporating the Cre recombinase plasmid into a positive ES cell clone and selecting for the appropriate clones. DNA from the resulting clones will be screened by the investigator and ES cells from clones with the desired gene orientation will be injected into blastocysts.
Karyotyping
Chromosome counting will be performed on positive ES cell clones prior to microinjection to ensure a euploid karyotype. If necessary, Spectral Karyotyping (SKY) can also be performed to further characterize chromosomal aberrations.
Contract Research Services
We are always interested in discussing unique animal model experiments. Please do not hesitate to contact us with those observations that might make interesting research projects. We may be able to explore collaborative arrangements and discuss experimental design.