Generation of transgenic mice
Transgenic mice are produced by pronuclear injection of a user-provided gene construct into mouse zygotes, which develop to term after surgical transfer into recipient female mice. Mice in which the transgene has integrated into the genome are identified by user's laboratory using PCR of tail DNA. Positive “founder” mice are transferred to the investigator for breeding and analysis. The TMF’s expertise includes injection of large DNA constructs, e.g. Bacterial Artificial Chromosome (BAC) DNA constructs.
Generation of knockin/knockout mice
Gene-targeted mice are produced by coinjection of CRISPR reagents together with a homologous recombination substrate into mouse zygotes. Site-specific CRISPR gRNAs and constructs are designed either by the TMF or by the user. 10-60% (depending on the target site) of resulting founder mice typically contain a mutant allele at the target locus, although this is not guaranteed. The traditional targeting approach involving transfer of mutant ES cells into mouse blastocysts is also still available to users.
Mouse embryo cryopreservation
Mouse lines can be cryopreserved for facility users, to protect irreplaceable mouse lines in the event of a catastrophic loss, and preserve mouse lines that are not in current use. For this purpose, two-day embryos are isolated from hormone-treated donor females that have been mated with the appropriate transgenic or knockout males, typically in the investigator’s colony. Fifty to one hundred embryos per strain are collected and frozen in two to three separate aliquots. Frozen embryos are stored in a liquid N2 tank maintained by the Facility. The TMF also performs thawing and re-implantation of embryos at the request of users, and has a 100% success rate in reviving lines frozen by the TMF.
The TMF provides support and advice for investigators during all phases of knockout and transgenic mouse projects, including targeting strategy and vector design.