Create your Transgenic Mouse or Rat in four easy steps:
Please contact Kathy Krentz (firstname.lastname@example.org) to schedule an initial meeting.
1. Guidelines for Transgene Vector Design
These common transgene features are often incorporated into the transgene vector. Vectors are available commercially.
a. promoter with a known cellular expression profile
b. gene or coding sequence of choice
c. a fragment containing polyadenylation signal
d. well characterized restriction sites that will allow the isolation of the transgenic fragment from plasmid backbone.
2. Purification of Transgene for Microinjection
Highly purified DNA is essential for successful transgenic production. Our facility will purify your digested DNA. Bring 30-50 µg digested DNA (150-200ul) to our lab with a gel picture clearly indicating which band we need to purify.
GEAM also has experience microinjecting BAC and PAC DNA. However, isolation methods are different.
Please contact us for the latest isolation procedure.
3. Microinjection of DNA into Oocytes
GEAM routinely microinjects transgenes into the pronucleus of C57BL/6J one-cell mouse embryos or Sprague Dawley rat embryos. GEAM has experience using other mouse strains such as FVB, NOD, NOD/Shiltj, ICR/HaJ, 129S1, and Btbr and rat strains F344, ACI, Lewis. Transgenics can be made in these strains but investigators must purchase the wild-type animals for embryo production. Other strains will also be considered.
Following the microinjections, the embryos are transferred into pseudopregnant recipients and pups born. At weaning, animals are ear-tagged and samples taken. Investigators are provided with tail DNA samples from each potential founder. The investigator must provide a copy of the genotyping results to the GEAM staff. The GEAM then will breed up to two founders when they reach breeding age to wild-type mates to produce one F 1 litter from each founder. The founder and litter are transferred to the investigator when the progeny are three weeks old. Any remaining founders will be shipped without breeding to the investigator.
ANIMAL AND EMBRYO SERVICES
Microinjection of CRISPR/Cas9 reagents into C57BL/6J embryos for the production of knock-out mice
Microinjection of CRISPR/Cas9 reagents into C57BL/6J embryos for the production of knock-in mice
Microinjection of transgenes into C57BL/6J embryos
Microinjection of purchased targeted ES cells into C57BL/6J blastocysts
Microinjection of CRISPR/Cas9 reagents into Sprague Dawley rat embryos for the production of knock-out rats
Microinjection of CRISPR/Cas9 reagents into Sprague Dawley rat embryos for the production of knock-in rats
Microinjection of transgenes into Sprague Dawley rat embryos