GEMM has been using the CRISPR technology to create genome-edited mice since June 2013.  We have created many mutant mice housing point mutations, exon deletions, and precise insertions of epitope tags, loxP, attP, EGFP and cre.  Our fees for CRISPR-based germline modifications are very competitive.

Transgene Injection Information

Transgenic mice are constructed by injecting cloned DNA into fertilized mouse eggs; those eggs that survive are then implanted in foster females to develop to term. The gestation period of the mouse is 19-21 days. Pups are ready for weaning at 3-4 weeks of age, and reach sexual maturity at 6 weeks (females) to 8 weeks (males). The minimum elapsed time between injection of the construct and readiness for breeding of the transgenic founders is 9 to 11 weeks. For most experimental purposes researchers will want to use offspring of a transgenic animal, rather than the founder animal itself.Several factors can affect the production of transgenic mice. For example, the purity of the DNA construct to be injected is very important to avoid toxic effects to the eggs, and the DNA must be at the proper concentration. The DNA construct must be purified away from vector sequences. Also, some expression constructs (e.g. beta-actin-MyoD) may have activities that are lethal for the transgenic embryos. Another caveat is that if transgene integration occurs after the first round of DNA replication in the single fertilized mouse egg, the mouse will be mosaic for the transgene, i.e. only a subset of cells will carry the transgene, and if the germ cells don’t carry the transgene, it will not transmit to the offspring. We will make every reasonable effort to make transgenic mice with your construct, but no facility can guarantee success with every construct.The core will isolate the transgene DNA from the DNA constructs provided by the investigator, then microinject this into approximately 300 fertilized eggs, transfer the embryos into foster females and monitor the pregnancies. The core will perform the tail biopsies and tag the pups, then transfer the pups and biopsies to the investigator. The investigator will perform DNA isolation from the tail clips, and analyze the DNA by PCR and/or Southern blotting in a timely manner. Pups, including potential transgenic founder animals, will be transferred to the care of the investigator upon weaning (3 weeks of age), who will arrange appropriate Vivarium housing and breeding for them outside of our facility.If the studies on tail biopsy DNA (with adequate controls) fail to demonstrate the presence of at least three transgenic animals, we will reinject the construct at no additional cost to the investigator. If no transgenic animal is detected after the second set of injections, we will hold a meeting to discuss further plans, such as re-purification of the DNA construct, or repeat injections and analysis of potential transgenic embryos if the DNA construct may result in embryonic lethality.

The investigator should provide a high purity DNA construct, the core will further purify the transgene away from the vector. The investigator must purify DNA from the tail biopsies and analyze the samples to determine which mice carry the transgene, in a timely manner. The pups, including the transgenic founder mice, will be transferred out of the facility to the care of the investigator upon weaning.

Transgenes should include promoter, enhancer, gene to be expressed, splice donor and acceptor and intron sequences, and termination/polyadenylation sequences. Transgenes must be excised from the bacterial plasmid sequences in order to be expressed in mice.

Congenic Mouse

Speed Congenics – Traditional development of a congenic strain, which involves serial backcrossing to transfer a gene (allele) from a donor strain to a recipient inbred strain, requires 2.5 to 3 years to complete 10 generations of backcrossing (N10).  In contrast, speed congenic strain development relies on microsatellite marker difference (simple sequence length polymorphisms, SSLPs) or SNPs between strains by selective breeding of progeny with a higher percentage of recipient genome, thereby shortening the generations of backcrossing to 5 (N5, 1.25 to 1.5 years).  In addition, genome scanning can be performed with a panel of mapped SSLP makers or SNPs for not only (1) introgressing target loci onto selected inbred backgrounds, but also (2) linkage analysis and (3) characterizing genetic backgrounds. Please contact GEMM if you need this service.

Gene Targeted Mouse

The core outsources the gene targeting projects.  Once appropriately targeting ES cell clones have been identified, they should be expanded and aliquots refrozen, tested by karyotyping and for mycoplasma, and the core will schedule blastocyst injections with the desired number of clones in consultation with the investigator.


Embryonic Stem Cell

Gene targeting in mice has been accomplished traditionally with embryonic stem cells derived from 129 strains.  Because of their genetic variability, poor reproductive performance and abnormal behaviors, the 129 knockout mice are therefore preferably backcrossed to C57BL/6 mice.  C57BL/6 is a reference strain of the mouse genome, many mutants are maintained on this background and widely used in genetic, immunological and behavioral studies, which provide significant advantages of producing knockout mice directly in the C57BL/6 strain.  Although the first C57BL/6 embryonic stem cell line was derived as early as 1991, a few lines have been successfully used in the past decade.

The core has derived multiple ES cell lines from the following mouse strains: C57BL/6, C57BL/6J-Tyrc-2J and NZM2328.  The C57BL/6 and C57BL/6J-Tyrc-2J ES cells lines can be used for gene targeting directly onto the C57BL/6 background, therefore circumventing the need for backcrossing.  NZM2328 is a congenic strain and a valuable mouse model for systemic lupus erythematosus (SLE) developed at UVa.  The ES cells from NZM2328 will facilitate direct exploration of SLE susceptible genes in this model strain.

The core also has stocks of popular germline competent 129 ES cell lines R1, CJ7, W4, RW4 and C57BL/6 ES cell lines.  Appropriate MTAs are required before we can fulfill the requests for these ES cell lines.

Murine Embryonic Stem Cell Resource

Mouse ES cell lines used in GEMM
129 strains – New nomenclature
R1 (Progeny of 129X1/SvJ and 129S1/Sv-+p +Tyr-c MgfSl-J/+) – Nagy A et al, Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8424-8
CJ7 (129S3/SvImJ-+p +Tyr-c +Mgf-SlJ/J) – Swiateck PJ and Gridley T, Genes Dev. 1993 Nov;7(11):2071-84
– matching isogenic BAC library CT7 from California Institute of Technology and sold by Invitrogen
W4 (129S6/SVEvTac) –
– matching isogenic Lambda library from Stratagene (now Agilent) or matching isogenic BAC library RP22 from Roswell Park Cancer Institute and sold by Invitrogen
RW4 (129X1/SvJ) – Heusel JW et al, Cell 1994 Mar 25;76(6):977-87

4A4 (C57BL/6, core-derived) – Reference available.

B1 (C57BL/6J-Tyrc-2J, core-derived) – Reference available.

Bruce4 (Thy1.1 congenic C57BL/6 strain) – Kontgen F et al, Int Immunol. 1993 Aug;5(8):957-64.
#693 (C57BL/6) –
– matching isogenic BAC library RP23 from Roswell Park Cancer Institute and sold by Invitrogen

Mouse ES cell lines available from commercial sources
Stem Cell Center –

ES cell karyotyping
Applied Genetics Laboratories –


Cryopreservation of gametes or embryos from scientifically valuable mice has become increasingly important. 1st, once the cryopreserved gametes or embryos are frozen and maintained under liquid nitrogen, they can theoretically be held indefinitely without any adverse effects. The first groups of mouse embryos were frozen in 1972 and thawed 25 years later in 1997 to produce live pups in The Jackson Lab. 2nd, the gametes or embryos stored in liquid nitrogen offer a safe way to preserve lines which are potentially but not currently useful, saving space and the expense associated with keeping a breeding colony. 3rd, during the process of gamete and embryo collection most pathogenic organisms are excluded, similar to other methods used to rederive lines of mice.

Once you have identified a strain for embryo cryopreservation, you should test a group of 6 adult (3 to 6 months) homozygous (preferred) males to determine their fertility. Using experienced males who have already impregnated females and rested for two weeks gets the project off to a quick start and is less costly than paying us to find out that particular males are subfertile or unfertile.

In order for us to use your males, they will have to be transferred to our animal protocol (3520) and room (OMS 1843), but the per diem will be charged to your account.  You should send an email to CCM director to ask for a permission for mouse transfer followed by submitting a CCM transfer form.

We normally purchase on your account females of an appropriate strain to present to your males, resulting in heterozygous embryos for freezing.  If your strain requires that we use your females, you should be able to provide 3 week-olds, one per male, for 12-18 females depending on how many embryos you want to freeze.  We will superovulate the females and present one female to each male once a week, then harvest embryos at the morula stage for the freezing procedure until the requested number of embryos has been preserved.

When we have frozen the requested number of embryos, we will thaw one straw to check for viability and we will either return the males to you or euthanize them per your instruction.