Joslin Genome Editing Core
The Genome Editing Core (GEC) provides DRC investigators with resources to manipulate the genome of human cell lines and laboratory mice with the aim of studying the genetics underlying diabetes and its complications. To facilitate these genetic studies, the GEC maintains a centralized facility for the generation and propagation of patient-derived induced Pluripotent Stem cells (iPSC). iPS cells hold tremendous potential for disease modeling applications as these cell lines retain the same genetic make-up as the patient's somatic cells targeted for reprogramming. The addition of gene editing tools (such as CRISPR/Cas 9) with iPSCs enables the study of genetic perturbations within a fixed genetic background, overcoming limitations inherent to studies based on genetically heterogeneous patient samples. The GEC also maintains a facility for the generation of transgenic mouse models to aid in studying the genetic mechanisms underlying diabetes in vivo. The mouse modeling facility uses CRISPR/Cas9 or lentiviral RNAi constructs to introduce genome modifications to a variety of mouse lines in order to establish new models of the disease within a living mammalian system.
Induced Pluripotent Stem Cell Core (IPSC) Services
Induced pluripotent stem cells (iPS cells), generated by transcription factor-dependent nuclear reprogramming of differentiated somatic cells, are pluripotent stem cell lines that can be propagated indefinitely in culture and maintain the potential to differentiate into any cell type in the body. As iPS cells retain the same genetic make-up as the somatic cell targeted for reprogramming, these cells hold tremendous promise for uncovering novel genetic and biochemical factors that underlie diseases with complex and poorly understood genetic influences, such as diabetes. The newly established iPS Core maintains a centralized facility for the reliable and consistent generation and propagation of reprogrammed iPS cells for use in cutting-edge research into the molecular and cellular pathologies underlying diabetes and its complications.
IPSC Services Offered
- Non-integrative Reprogramming. Human iPSC lines will be generated from cultured fibroblasts, peripheral blood, or other relevant cell types using Sendai virus or episomal plasmid transfection.
- Genome Editing Services within iPS lines. Genetic modifications including gene knock-outs and gene corrections are generated using CRISPR/Cas9 gene-editing tools.
- Validation and quality control of iPS lines. Our core services include pluripotency characterization (including Flow Cytometry, Real-Time PCR, and Teratoma Formation analysis), Karyotyping, and Mycoplasma testing.
- iPS cell line expansion, banking, and distribution.
- Discounted iPS culture reagents. Joslin users can purchase discounted iPS culture reagents from a variety of vendors directly through Joslin's Cortex Website.
- Training in iPS cell culture and subsequent use of core facilities for self run studies. Workshops for iPS culture training are scheduled annually. For information on protocols and techniques used by the core, please visit the Stembook Website.
- Genome Editing Services. Our core has recently been able to provide genome modification services using the CRISPR/Cas9 system in iPSC and other cell lines. Investigators should contact core personnel to set up a meeting to discuss details of their project.
Mouse Genome Editing (GEC) Services
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.
GEC Services Offered
- 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 vitro assays will be provided when requested.