ES Cell & Embryo Services
The Transgenic Facility provides services for the production of genetically altered mice using embryonic stem cell technology. Before any work on a project can begin, a DUIACAUC protocol must be approved. Also a Service Application for ES Targeting should be filled out and submitted to the facility. If a PI is submitting ES clones that have already been targeted, please use this form:
The project leader should carefully design a targeting vector using a 129 mouse library. The facility has used the R1 ES cell line since 1996, but we are in the process of switching to the Sv/Ev ES cell pedigree. PCR generated constructs have not worked well in our hands for targeting, giving very low percentage, or—disappointingly—zero targeting. The Transgenic Facility advises that constructs have homology to the locus above 7 Kb in length. Longer is better. The facility relies on an initial PCR screening of picked clones to identify potential positives. The screening utilizes a PCR product derived from the neomycine sequence through the short arm out to the region of homology beyond the targeting construct. We ask investigators to find primers that work under our screening conditions for cocktail formulation and PCR program. For this reason, the short should not be more than about 1.6 Kb, as the PCR product should be under 2 Kb. We ask that a control mock construct and primers be submitted to us after testing by the project leader along with a photograph of serial dilution to detect femtogram amounts of DNA in genomic background. For discussion of details on the test protocol and PCR conditions contact Cheryl Bock by email to set up an appointment.
After positive clones are detected by the PCR screening strategy, the Facility will expand and freeze the clones. We will also grow samples of each clone on a 6-well size feederless plate to submit to the project leader for Southern analysis. It is critical to check each clone to assure that the mutation is as desired and planned, before we continue in the experiment to inject the cells to produce chimeric mice. A Southern blot also tells us critical information about mixed clones, something we cannot determine from normal PCR screening. At about ten days of age we will determine the percentage of chimerism of resulting pups. At this time we will fill out the transfer paperwork and submit this to DLAR. DLAR will transfer the chimeric mice to the project leader’s designated animal holding space, for your lab to breed and test. The Transgenic Facility must keep data about our experiments and we expect the project leader to report back to us on germline transmission for each chimera and clone.
Embryo Donors from PI Mouse Strain for Embryo Microinjection Experiments
The DCI Transgenic Shared Resource can only house mouse embryo donor strains from certified vendors. We currently have B6SJLF1/J, C57BL/6NHsd, C57BL/6J, FVB/NHsd available as embryo donors.
If a PI wants to use their own mouse strain as a donor, these are important points to consider:
Female mouse superovulation is a procedure that should be listed on the PI approved animal protocol. If this procedure is not currently on the approved protocol, it can be added as an amendment.
Mouse matings are set up 1:1 females to males. Superovulation efficiency depends on the mouse strain. A minimum of 10 pairs should be set up, but more, up to 20, will be OK. We can inject 200-350 embryos on one day.
Female mice generally work well at 8 weeks of age. Male mice should be proven fertile & not housed with a female within 1 week of the mating experiment. Male age can be 6 weeks to 9 months.
Hormone is supplied by LA Biomedical Research Institute at Harbor UCLA (National Hormone Peptide Program.) (45983 special order vendor at Buy@Duke)
Females are each dosed IP with 5 IU PMSG at about 3 PM day 1. On day 3, females are dosed IP with 5 IU HCG. (About 46-48 hours after PMSG injection) Females are immediately mated 1:1 with fertile males. On day 4, females are removed from male cages about 8:30 AM & plugs recorded. All females, plugged or not, are brought to the DCI Mouse Core in GSRB1 near 3038 for dissection. One cell embryos are harvested about 9 AM & injected the same day.