The Transgenic and ES Cell Core of the Washington University Diabetes Research Center (DRC) provides services for efficient and cost-effective development of genetically altered mouse models to elucidate the pathogenesis of diabetes and related metabolic disorders. Importantly, in vivo models allow 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.
Current molecular genetic techniques allow spatially and temporally restricted analysis of gene effects (both inactivation and over-expression) in mice, leading to previously unprecedented creativity and analytical resolution. The concepts involved in generating transgenic (Tg) or knockout/knockin (KO/KI) animals are straightforward. However, it is impractical and too expensive for most labs to maintain the requisite equipment, facilities, and highly skilled personnel for making genetically altered mice. Given the substantial demand by the Washington University community for assistance in the generation of these mouse models, the DRC Transgenic and ES Cell Core will continue to provide the necessary advice, expertise, facilities, equipment, and personnel to efficiently generate Tg and KO/KI mice for DRC members using state-of-the-art techniques, such as CRISPR/Cas9 nuclease-mediated genome editing in ES cells and in mouse zygotes. The Core will also assist in propagating poorly breeding animals, and in achieving transient Cre or FLP recombinase expression in zygotes by microinjection of expression plasmids. By making specialized, state-of-the-art services accessible to DRC investigators, the Core facilitates translation of basic research advances toward improvements in the diagnosis, treatment and cure of diabetes.
The Transgenic and ES Cell Core leverages three well established and highly functioning WUSM institutional Cores. The Mouse Embryonic Stem Cell Core (MESCC) and the Mouse Genetics Core (MGC) have proven track records of providing high quality service in efficiently generating targeted embryonic stem (ES) cells (the MESCC) and genetically manipulating mice (the MGC). During the past cycle, we added a new service for complex vector design by leveraging the WUSM institutional Transgenic Vectors Core (TVC).
Leadership and Contact
Professor of Medicine
660 South Euclid Avenue, Campus Box 8126
Advice for generation, genotyping, breeding, and maintenance of genetically altered mice
Consultations on general approaches and overall project design are provided by Dr. Miner.
Complex Vector and Gene Editing Design Consultation and Construction
For more complex vectors, such as modified bacterial artificial chromosomes (BACs), complicated knockins, or for gene editing using engineered nucleases (CRISP/Cas9 or TALENS), the Core not only supports consultations with the Core Director, Dr. Miner, and with Ms. Lewis, manager of the TVC, but also assists in complex vector or CRISPR/TALEN construction. Approaches supported include plasmid- and BAC-based transgenes and conditional and inducible knockins and knockouts generated by conventional or recombineering cloning techniques. The Core also designs and validates guide RNAs and DNA fragments for homology directed repair for CRISPR/Cas9-mediated gene editing approaches.
Mouse ES Cell Manipulation
The Core provides eight different lines of germline-proven ES cells (B6/Blu, B6/GFP, B6-13, EDJ22, LK-1, R1, SCC10, SWT16); grows and transfects ES cells 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. In addition, the Core will grow targeted ES cells obtained from the various entities affiliated with the International Knockout Mouse Consortium (IKMC), which is attempting to mutate all genes in the mouse genome (Knockout Mouse Project [KOMP] repository at UC Davis, the European Conditional Mouse Mutagenesis Program [EUCOMM], the North American Conditional Mouse Mutagenesis Project [NorCOMM, Canada], and the Texas A&M Institute for Genomic Medicine [TIGM]). Since the selection of euploid clones for injection increases the likelihood of obtaining germline contribution of the ES cells, the Core assists investigators in karyotyping successfully targeted clones.
Microinjection of DNA (plasmid fragment or BAC) into single-celled mouse embryos to generate conventional transgenic mice
The Core performs microinjections into the pronuclei of single-celled mouse embryos, using transgene DNA, purified away from plasmid vector sequences via a specific protocol provided by the Core, or using a whole BAC. We typically recommend injections over three days, but investigators can also choose single-day injection service on a prorated cost basis. For the 3-day injection service, a minimum of 300 embryos is injected, which typically results in the production of at least 2 transgenic founder animals. The Core offers three different mouse strains with which it has an excellent record of success: C57BL/6J, B6CBAF2/J, or FVB/N. Custom strains can be used for an additional charge. Embryos are incubated overnight, and those that progress to the 2-cell stage are surgically implanted into the oviducts of pseudopregnant females. Toes from potential founder animals are clipped at 5 days of age, DNA is extracted, and PCR genotyping is performed using a protocol optimized for very low concentrations of transgene DNA. Transgenic founders are transferred to the investigator’s own 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 with normal karyotypes are injected into 150-175 blastocysts or morulae (with the assistance of a laser to puncture the zona pellucida) over a 3-day period. While we typically recommend injections over three days, investigators can also choose single-day injection service on a prorated cost basis. Injected embryos are surgically 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 investigator’s colony for breeding and analysis.
Microinjection of nucleic acids into single-celled mouse embryos to generate CRISPR/Cas9- or TALEN-mediated knockout or gene-edited mice
mRNA encoding the Cas9 nuclease along with appropriate gene-specific guide RNAs are co-injected into the cytoplasm or pronucleus of single-celled embryos to induce mutations by non-homologous end joining of double stranded DNA breaks. We typically recommend injections over 1 or 2 days. For precise gene editing, donor oligonucleotides or larger DNA fragments containing specific mutations, loxP sites, or reporters such as EGFP are co-injected over 3 or 4 days to achieve homology-directed repair. For targeted insertion of genes at the widely expressed Rosa26 locus, TALENs that have proven efficient are co-injected with donor DNA for homology-directed recombination. Toe clips for molecular analysis by PCR and sequencing are provided to investigators so that they can determine whether the desired mutation or insertion has occurred.
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.
Rederivation/importation of mice/sperm from outside the institution:
For importing mice from other institutions that are not specific pathogen-free, the Core offers services to rederive the line by preimplantation embryo transfer. Alternatively, importing frozen sperm and performing IVF or importing preimplantation embryos, either frozen or at ambient temperature, and transferring to pseudopregnant females can reduce the overall cost of obtaining new mouse lines.
Transient expression of Cre or FLP recombinase in single-cell mouse embryos
To remove a selectable marker by expression of either Cre or FLP recombinase, the Core offers a service for microinjecting the relevant recombinase expression plasmid (Cre and FLP vectors are available in the MGC) into the nucleus for transient expression in single-cell mouse embryos carrying the allele to be recombined. Following injection, embryos are implanted and carried to term. After birth, the pups are screened for the successfully recombined allele and for the recombinase gene to confirm that the expression plasmid did not integrate into the genome.
Requests for Service
The institutional TVC, MESCC, and MGC function as recharge centers. The TVC, MESCC and MGC set WUSM chargebacks reflecting the cost of service after institutional support is taken into consideration. DRC Transgenic and ES Cell Core funding is used to offset these chargeback rates for DRC members carrying out studies of diabetes and related metabolic disorders. A portion of chargebacks for these services is billed to the Transgenic and ES Cell Core and the remainder to the investigator, effectively lessening the financial burden for DRC investigators who wish to develop mouse models for their diabetes studies.
|Table 2. Transgenic and ES Cell Core Chargebacks (subject to change)|
|General consultation||N/A||No charge|
|Complex vector or CRISPR/TALEN design consultation, construction||$60/hr||$40/hr|
|(100 hr max)|
|ES cell transfection and selection||$3,800||$2,000|
|Transgene injection (3 days)||$4,695||$1,695|
|ES Cell injection (3 days)||$4,695||$1,695|
|CRISPR/Cas9 or TALEN injections (3 days)||$4,695||$1,695|
|Recombinase expression in single-cell embryos (1day)||~$1,000||~$500|
|Ovary transplant or SPF rederivation||$215||$100|
Mutant Mouse Regional Resource Centers (MMRRC) supported by NIH MMRRC
Resources for existing CRE driver mouse lines
Resources for existing genetically modified mouse lines