Transgenic & Genetically Engineered Models

Research Administration Centers and Cores Transgenic & Genetically Engineered Models

Transgenic & Genetically Engineered Models

Directory Category: Institutional Core

Cell injectionDescription of Services

Located in the Kaul Building, TGEMS services include the production of mouse models using gene targeting and DNA and ES cell injection methods. We primarily use fertilized oocytes from C57BL/6 ("pure" B6) mice and B6XSJL (hybrid) strains, and 129 or B6 (black and albino) ES cells. Other strains are available upon request.

Additional services

Cryopreservation of embryos and sperm, embryo rederivation to produce pathogen-free mice, and assisted reproduction techniques (in vitro fertilization [IVF], superovulation, and embryo transfer).

Mouse photo courtesy of Dr. David Crawford, "G2E3 Inactivation Causes Early Embryonic Lethality"New Techniques

Perhaps the greatest resource currently available to researchers for the creation of "knockout" mouse models is the generation of libraries of random "genetrap" ES cell clones. Gene trapping is a cost effective method for producing ES cells harboring insertional mutations, most often into introns, which typically leads to a null allele of the "trapped" gene (see Figure 1). An additional advantage of gene trap vectors is that they incorporate the use of selectable reporter genes (e.g. lacZ gene), which aid in characterizing the tissue-specific expression of the trapped gene. The International GeneTrap Consortium (IGTC) maintains an updated database (www.genetrap.org) of available ES cell clones that can be identified through both BLAST sequence searches and keyword queries. For additional information or assistance in identifying a genetrap ES cell clone, please contact Dr. Lambert (or e-mail her your gene of interest).


genetrap
Figure 1. Diagram of genetrap process. Integration of a gene trap vector into an intron of a gene leads to the splice acceptor (SA) being utilized instead of the normal downstream exon. Therefore, the newly created targeted allele produces a lacZ fusion gene (which can be used to map expression of the gene), and most often a "null" allele due to disrupted downstream sequences. Shown at right are E14.5 day embryos heterozygous for a genetrap G2E3 gene allele (note: homozygous are embryonic lethal).

Gene knockout and editing by CRISPR-Cas9 system.

Guide design and preparation, Blastocyst assay to assess activity of guides, Establishment of genotyping strategy, Repair Template Design, Compatible with most efficient guide(s), Unique Restriction Site for downstream screening of founders, Pronuclear microinjection of 150 embryos, Transfer of embryos to pseudopregnant recipients, Optional screening of founder animals, Transfer of founder litters at weaning.