The goal of the Washington University Diabetes Research Center Transgenic and ES Cell Core is to assist investigators in the efficient and cost-effective development of genetically-altered murine models to elucidate the pathogenesis of diabetes and its complications and to explore related areas of endocrinology and metabolism. These novel animal models serve as an important avenue for pre-clinical studies that will ultimately provide the basis for translation of basic research advances into clinical practice. Importantly, in vivo models will allow the analysis of gene function in the context of complex intercellular and integrative systemic responses needed for the study of diabetes, a polygenic disorder involving multiple tissue types and environmental influences.
Jeffrey H. Miner, Ph.D.
Professor of Medicine
Internal Medicine, Renal
Mailing Address: Campus Box 8126
Office Phone: (314) 362-8235
Fax: (314) 362-8237
Advice for the generation, genotyping, breeding, and maintenance of genetically altered mice
Consultations on general approaches and overall project design are provided by Dr. Miner.
Complex Vector Design Consultation
For complex vectors (e.g., modified bacterial artificial chromosomes (BACs), complicated knockins), the DRC subsidizes consultations and services of the Washington University Transgenic Vector Core for production of transgenes (plasmid- and BAC-based) and targeting vectors (including conditional and inducible knockins and knockouts), design of recombineering approaches and PCR screening assays.
ES Cell Transfection
The DRC Subsidizes use of the Siteman Cancer Center ES Cell Core, which provides six different lines of germline-proven ES cells; grows and transfects them by electroporation; picks and expands drug-resistant colonies; freezes colonies and retains them while they are screened by the investigator for homologous recombination or transgene integration/expression; and expands positive colonies for further analysis and/or for injection into blastocysts or morulae. The Core will also grow targeted ES cells obtained from entities affiliated with the International Knockout Mouse Consortium (IKMC) and assists investigators in karyotyping successfully targeted clones.
Microinjection of DNA (plasmid fragment or BAC) into single-celled mouse embryos to generate conventional transgenic mice
Transgene DNA or a whole BAC is microinjected into the pronuclei of single-celled mouse embryos. Embryos are incubated overnight, and those that progress to the 2-cell stage are implanted into the oviducts of pseudopregnant females. DNA is extracted from tail tips of the offspring and subjected to PCR genotyping by the MGC. Transgenic founders are transferred to the investigator’s colony for propagation and analysis.
Conventional or laser-assisted injection of modified embryonic stem cells into blastocysts or 8-cell embryos (morulae) to make knockout, knockin, or transgenic mice
Successfully targeted ES cells are injected into 150-175 blastocysts or morulae (with the assistance of a laser to puncture the zona pellucida). Injected embryos are implanted into the uterine horns of pseudopregnant females. This usually results in the birth of 30 to 50 mice, about 6 of which have high percentage ES cell contribution and good probability of germline contribution. High percentage chimeras are transferred to the colony of the investigator for breeding and analysis.
Generation of cogenic lines by marker-assisted breeding (speed cogenics)
Genomes of several backcross mice at each generation are scanned to find the ones with better than average contribution from the recipient background. The ones with the highest contribution are selected for the subsequent backcross to obtain the next generation. By this approach, only 5 backcross generations are usually required to attain congenic status. Users of the speed congenic service transfer two males containing the genome alteration to the Mouse Genetics Core. Core personnel set up matings to the recipient strain, purify DNA from tail clips of the offspring, and send the DNA to a facility that performs the genome scans using SNP (single nucleotide polymorphism) chips.
To prevent the loss of crucial mouse lines that breed poorly, the MGC can perform either ovary transplantation and superovulation with nonproductive females, or in vitro fertilization (IVF) with sperm from nonproductive males.
Cryogenic sperm preservation
The MGC can freeze down sperm for multiple tries at IVF and for long-term preservation of unique lines
2. Complex vector
design and consultation
3. ES cell transfection
4. Transgene injection
5. ES cell injection
6. Speed congenic
7. Assisted Reproduction
8. Rederivation / Importation service
9. Transient expression of recombinase
10. Embryo Aggregations
(per 3 day project)
(per 3 day project)
$150 – $712
$300 – $600
Investigators wishing to use services of the DRC Transgenic and ES cell core should download, complete, and send the following forms to Dr. Miner at firstname.lastname@example.org