This Core provides cutting-edge technology to assay gene function both in living animals and in cell culture. The Functional Genomics and Genetics Shared Resource offers assistance with the construction and management of genetically altered mouse strains, as well as RNAi technologies and CRISPR reagents. For pricing information or other questions contact the Resource Manager, Ken Chang.
The Core generates transgenic or chimeric mice, and offers cryopreservation of both mouse embryos and sperm for storage and management of transgenic mouse lines. In addition to these standard services, the facility assists investigators in experiments that require sophisticated embryo manipulation or cell/embryo microinjection techniques. Reagents include:
Transgenic Mouse Services
- Production of transgenic mice by pronuclear injection
- Generation of chimeric mice by injection of genetically altered ES cells
Cell Culture Services
- Gene targeting in embryonic stem cells
- Derivation of ES cell lines from transgenic or mutant mice
- Maintenance of ES cell line stocks for use in gene targeting experiments
- Karyotyping of ES cells
- In vitro fertilization (IVF)
- Intracytoplasmic sperm injection (ICSI)
- Embryo and sperm cryopreservation
- Recovery of frozen embryos
- Rederivation of SPF mouse lines
- Consulting and technical assistance
This Core offers both RNAi and CRISPR screening technology. We provide guidance, reagents, technical support, and data analysis to enable researchers to perform shRNA and CRISPR screens. Investigators interested in screening and/or obtaining reagents are encouraged to set up a consultation with Ken Chang. Services include:
- Consultation. Initial and regular meetings to discuss the overall needs, services, and research objectives as well as optimization, and troubleshooting.
- Genetic screens using RNAi and CRISPR libraries. Assisted pooled shRNA and CRISPR screening is offered at sub- and full-genome scale.
- Access to shRNA clones and libraries. shRNA libraries are available for silencing gene expression in mammalian genomes. shRNA clones are available either as individually arrayed and pooled glycerol stocks, or as purified DNA, which can be used for virus production or transfection-based assays. Focused shRNA collections to target kinase, phosphatase, epigenetic, ‘druggable’, or custom genes for human and mouse genomes are also available to researchers.
- Access to CRISPR clones and libraries. Domain-based, pooled CRISPR libraries targeting genes of functional groups, including epigenetic, kinases, phosphatases, transcriptional factors, from human and mouse genomes, are available. The Resource will also provide the service of constructing individual sgRNA expressing clones in lentiviral vectors to investigators.
- Virus production of shRNA and CRISPR collections
- Design and construction of high depth custom shRNA and CRISPR libraries. Custom shRNA libraries can be designed using the shERWOOD algorithm, which computationally predicts highly potent shRNA sequences. CRISPR sgRNAs libraries, using an algorithm designed by the Vakoc laboratory can produce negative selection phenotypes that are an order of magnitude stronger than phenotypes derived from using conventional CRISPR libraries, which are designed for mutagenesis of 5’ exons.
- Access to shRNA expression vectors. The Shared Resource has tetracycline (Tet)-inducible viral vectors containing different markers capable of tracking viral transduction and shRNA expression. A variety of constitutive promoters (EF1-alpha, hCMV, mCMV, mPGK, SFFV, CAGS, MSCV-LTR, and RSV) are available for inducing gene silencing in multiple cell types. Cloning services are available for construction of custom shRNA libraries tailored for specialized applications.
- Access to other biological resources. Small RNA resources (siRNA, miRNA, dsRNA, and compound libraries) and human and mouse full-length cDNAs and ORFs based on the MGC collections (NIH) are available, representing approximately 15,000 genes of the human and mouse genomes.
- Equipment Access. Equipment is available for screening, including: MacsQuant FACS analyzer; BIOMEK Robotic liquid handler; Perkin Elmer Enspire plate reader; Illimina Miseq sequencer; Nikon Fluorescence microsope (with z-stacking), Bio-Rad CFX96 Real time PCR system, and cell culture hoods/incubators.
Generation of constitutive or conditional gene knockouts may be facilitated by use of available libraries of gene targeting vectors or interfering constructs. Such resources greatly speed up generation of mouse models with defined germline mutations.