Molecular Genetics Core

The broad specific aims of this Core (Richard Flavell, PI) are to enable diabetes-related research through the generation of unique, genetically-modified mutant mouse models.

We currently provide the following three principal services contained within three distinct, but tightly interrelated Subcores:

1. The Molecular sub-core generates custom designed DNA and RNA constructs for genetic modification in mice and provides PCR genotyping and quantitation and localization of gene expression;
2. The Gene Targeting sub-core generates genetically modified mouse strains on a number of genetic backgrounds; and
3. The Diabetic Mouse Breeding sub-core maintains and interbreeds key strains of mutant and/or genetically modified mice and distributes them to diabetes researchers within the DRC, including novel humanized models and genetically modified NOD strains generated by the Molecular and Gene Targeting sub-cores.

While the current structure of the Molecular Genetic Mouse Core remains ideally suited to the generation and characterization of animal models, our methodology has undergone a transformational shift in the last funding period with the development of CRISPR/Cas9 gene editing technology, which has, to a great extent, replaced our prior methods for generating animal models.The goal of the Molecular Genetics Core is to provide DRC members and their collaborators with unique rodent models that are not easily duplicated by commercial facilities.

The main purpose of the Gene Targeting sub-core is to make available a service facility that will ensure the capacity of DRC members to produce genetically targeted mice. Not only does this Core facility make available specialized technologies, it also results in a great reduction in expense and effort by eliminating the duplication of highly technical skills that require expensive specialized equipment and animal facilities. The availability of genetically modified, inbred, strains of mice is central to our ability to understand the physiologic and immunologic mechanisms underlying diabetes.

The unique contribution to the DRC of this sub-core is its ability to create the next generation of humanized mice. We provide core services for the generation of genetically-modified strain hybrid F2 mice, inbred C57BL/6, and NOD mice. It is noteworthy that direct targeting of NOD mouse genes, whether by conventional transgenesis or CRISPR, is a technique mastered by only a few institutions in the nation and our Core was the first facility that was able to target the NOD mouse directly. The Diabetic Mouse Breeding Subcore maintains a colony of NOD mice from which we generate male stud mice and produce 3 wk-old female mice for egg production. We also continue to offer the generation of chimeric mice for genetically manipulated embryonal stem cells from 129, C57BL/6, and NODx129 F1 ES cells, although these services have been largely superseded by CRISPR approaches. Finally, we provide cryopreservation of embryos or sperm, microinjection of sperm and in vitro fertilization. This Subcore, together with Molecular and Diabetic Mouse Breeding Subcores, is currently undertaking a major effort to generate the humanized MISTRG humanized model on the NOD genetic background for T1D studies.

The Molecular sub-core supports three populations of diabetes investigators at Yale, each with a distinct set of requirements:

1. Investigators from clinical or other non-molecular genetic disciplines who need to have animal models created and/or characterized for their research,
2. Investigators who are attempting to learn new techniques and need step-by-step guidance or training to become self-sufficient, and
3. Experienced investigators who encounter a specific need to have a specialized assay or process performed for which they are not equipped.

In an effort to best meet such needs of our members, we have continued to adjust our resources and goals accordingly and currently provide the following:

1. critical services
2. technical assistance
3. training
4. equipment and resources centered around two principal areas of molecular genetics expertise, the generation of gene expression and gene targeting constructs for the creation of animal or cell culture models (principally via CRISPR/Cas9 approaches), and the analysis of gene expression in animal models, by quantitative RT-PCR and in situ hybridization, PCR-based in situ hybridization, immunohistochemistry and specialized staining.

• Tissue-specific transgenics
• Inducible/ repressible transgenics (tTA, rtTA)
• Whole animal constitutive gene knock-outs (CRISPR)
• Conditional knock-outs (Cre-loxP, FLP-frt, recombination site mutants via CRISPR)
• Inducible knock-outs (CreER, CrePR via CRISPR)
• Gene knock-ins (CRISPR-mediated mutations, tags, reporters (IRES or 2A), coding region in-frame insertions or deletions, CDS or whole gene replacements)
• Activatable knock-ins (lox-STOP-lox via CRISPR)
• BAC recombineering (gene additions, modifications, replacements)
• Additional services include: Genotyping designs, mutant strain genetic characterization, genomic localization (inverse PCR), allelic discrimination (homozygosity determination), and assay of gene expression by quantitative RT-PCR

For inquiries or project submissions, please contact William Philbrick (william.philbrick@yale.edu)