This Core enables investigators to manipulate the genome of laboratory mice in order to generate the most relevant experimental models to understand the genetics and mechanisms underlying diabetes and its complications. The GEC blends Joslin's research expertise in mouse models for diabetes and the generation of genetically modified mice.
The GEC will support the production of genetically modified mice, tailored to investigators' experimental questions by performing pronuclear and intracytoplasmic microinjections. If necessary, the Core can also provide lentiviral transgenesis services for generating mice that carry transgenes mediating inducible or constitutive silencing of target genes of interest by RNAi. The primary method of genetic modification is the CRISPR/Cas9 complex.
The Core will perform mouse genome editing using a variety of targeted edits made possible through CRISPR/Cas9. This includes creating knockout mice by generating a random mutation following a double stranded break (DSB) and non-homologous endpoint joining (NHEJ). For more specific modifications, the use of DNA templates can effect sequence insertion or replacement for knock-in models, for example. Other methods of influencing gene activation or repression are available and can be used upon request.
- Generation of CRISPR-modified mice. GEC director and staff will aid in the design and preparation of guide RNA and DNA templates for sequence deletion or replacement. Investigator-provided materials will be combined with GEC-provided Cas9 mRNA or protein and injected in GEC-provided C57BL/6 and NOD zygotes. Investigators may provide a different strain of mouse, if necessary.
- Generation of lentiviral shRNA transgenic mice. Spearheaded by Dr. Kissler's expertise with the generation of knockdown mice, the GEC will make available a diverse repertoire of lentiviral constructs for constitutive and doxycycline-inducible gene silencing. Following the standardized preparation of high-titer virus by the investigator's laboratory, the Core will microinject zygotes with lentivirus.
- Consultation on design of gene-modified mice. The GEC will provide advice on construct design for both CRISPR-Cas9 genome editing and lentiviral RNAi. Consultations with investigators will ensure that all researchers use standardized, GEC-approved protocols for preparing injection reagents.
- Consultation on CRISPR gRNA design and testing. The GEC will share its experience with CRISPR/Cas9 to aid in the design of efficient, on-target sequences and of strategies for sequence replacement or gene knock-in. Advice on the use of certain in vitroassays will be provided when requested.