The Core will assist investigators in expansion of mouse ES cells clones purchased from repositories, obtained from researchers at other institutions, or targeted in their own lab. We will expand cells and send sample for murine virus testing prior to injection. Microinjection will be processed, and investigators will receive potential chimeras and breed to germline transmission. For 129 ES cells, C57BL/6J blastocysts generated via super ovulation will be used to generate chimera founder mice; for C57BL/6N ES cells, FVB 8-cell embryos generated via super ovulation will be used to generate chimera founder mice.
What Will Happen
- The investigator will submit a request form through iLab with a detailed gene targeting strategy and proven screening protocol. An account and IACUC number must be provided at this time.
- Investigator will bring frozen vial(s) of ES cell clones to the core along with culture protocol and recipes.
- The core will review investigator’s request and schedule a date to thaw clone.
- The core will expand clone in the appropriate media and on feeder cells if needed, split into five vials, freeze and store in liquid nitrogen.
- The core will submit ES cell sample to RADIL laboratories for murine virus testing. Virus testing is mandatory by the BCM Center for Comparative Medicine prior to ES cells being microinjected. If ES cells have also not been karyotyped, the Core can also coordinate cells to be sent out for chromosome counting.A cell pellet will be given to investigator should he/she need to do further genotyping of cells.
- The core will schedule microinjection of cells into blastocysts/8-cell embryos after receiving murine virus test results. The core will use appropriate recipient embryos for coat color identification of chimeras and offspring. Examples:
- 129 (agouti) ES cells: B6 embryos
- B6 (agouti) ES cells: FVB 8-cell embryos
- B6 (black/non-agouti) ES cells: FVB 8-cell embryos
- Superovulation (129 and B6 ES cells) will be used to produce embryos.
- The core will microinject 45 embryos per clone.
- The core will perform microinjections of up to 2 clones per request.
- Additional clones will incur extra cost.
- Dams will be allowed to litter in core space. Litters will be transferred to investigator space at two weeks of age. The number, sex, and percent ES cell coat color contribution of chimeras will be indicated.
- Investigator will breed chimera and confirm germline transmission of gene.
What is Expected
- Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
- ES cell expansion and freezing
- ES cell virus testing
- Blastocyst injections
- Chimeric animals with coat color contribution from the injected ES cell clones ranging from 10%-100%. Coat color contributions does not necessarily represent ES cell contribution to cell lineages in all tissues, in particular the germline. However, males with the highest amount of coat color contribution from the ES cells should be prioritized for breeding and as many males as possible should be bred.
- The number of chimeric animals produced and the range of coat color contribution from an ES cell clone can vary widely and can depend on the pluripotent potential of the ES cell clone and potentially the targeted gene.
- Germline transmission of the targeted allele can require several rounds of breeding attempts. Transmission efficiency is dependent on overall ES cell contribution to the germ cell population (influenced by chromosome stability and pluripotent capacity). Good coat color contribution is not a guarantee of germline transmission. Moreover, as targeting in ES cells almost always results in only one modified allele (heterozygosity), coat color and not the targeted allele can be transmitted. Targeted gene function can affect allele transmission.
- Female chimeras should not be bred. They will often be sterile and male offspring will be sterile.
- There is no guarantee that chimeric mice will be produced or that chimeras will breed. Additional microinjections with the same ES cell clones or different clones will be done at 100% of cost.