Investigator submits a targeting strategy and demonstrates a proven screening protocol. After approval of investigator’s approach, the core will initiate electroporation in mouse ES cells. Investigator-screened targeted clones will be expanded by the core, sent for chromosome counts, and microinjected into blastocysts for chimera production. Investigators will receive potential chimeras and breed to germline transmission. All projects are scheduled through iLab on a first come first serve basis.
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. The ES cell line to be used is selected at this time. ES cell lines available for targeting are:
- AB2.2: 129/SvEvBrd [Hprtb-m2]
- E14Tg2a: 129/OlaHsd [Hprtb-m3] (feeder free)
- JM8.F6: C57BL6/Ntac
- JM8.N4: C57BL6/Ntac
- The core will review investigator’s targeting strategy and approve project or make recommendations.
- The investigator will generate their targeting construct. The GERM Core does not provide construct generation as a service.
- The core will schedule the project and prepare cells for electroporation.
- The investigator will prepare linearized or circular vector for electroporation and bring to the core as pellet in 100% ethanol. Projects involving B6 ES cells require 250ug of DNA. Projects involving 129 ES cells require 150ug of DNA.
- The core will prepare mouse ES cells for electroporation, perform electroporation, and select/isolate clones. The core will pick up to three 96 well plates of clones. Additional plates will incur extra cost. Triplicates of each plate will be generated: two copies of each plate will be prepared as lysate and given to investigator for targeting screen; one copy will be stored by the core at -80C for expansion.
- Investigators will perform all ES cell genotyping and must return results to the core no later than three months after receiving lysate plates. The GERM Core does not provide ES cell genotyping services.
- The Core will expand up to 10 targeted clones. Clones will be graded according to culture morphology in order to select three clones to send out for chromosome counts and finally microinjection. ES cell clones must have a minimum of 50% euploid nuclei for microinjections.
- The Core will provide investigator a DNA pellet for confirmation of expanded clone targeting; the investigator will notify that the expanded clones harbor a targeted allele.
- The core will schedule microinjection of ES cells into blastocysts/8-cell embryos after chromosome counts are determined. 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 generate embryos for microinjection.
- The core will microinject 45 embryos per clone.
- The core will microinject up to 2 clones for 129-derived and up to 3 clones for B6-derived ES cells.
- 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:
- Freezing of 96 well plates of picked ES cell clones
- Expansion and freezing of selected ES cell clones
- ES cell chromosome counting
- Blastocyst injection
- If targeted ES cell clones are not identified, the electroporation will be repeated at 50% cost.
- 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.