The Genetically Altered Mouse Core (GAMC) provides a centralized service to efficiently produce infection-free transgenic, chimeric, and genome-edited strains of mice. Among the services provided by the GAMC are generation of genetically altered mice via direct genome editing (CRISPR/Cas9, and less frequently Zn finger and TALEN technologies), by DNA microinjections into fertilized oocyte to create transgenic lines, or by generation of chimeric mice via embryonic stem cell injection into blastocysts. The Genetically Altered Mouse Core also carries out embryo re-derivation, embryo and sperm cryopreservation, in vitro fertilization (IVF), and centralized cryopreservation storage. The GAMC uses state-of-the-art laser conditioning of the zona to facilitate IVF and has intracytoplasmic sperm injection capability on-line to complement IVF services. Newly developed services include the major expansion of cryopreservation services with corresponding expansion of the cryopreservation facility, integration of CRISPR/Cas9 direct genome modifications with a newly established Perelman School of Medicine CRISPR core, and electroporation of DNA and RNA into embryos to increase throughput and decrease wait time for GAMC services. The Facility is led by Dr. Douglas Epstein, Professor of Genetics.
The GAMC provides comprehensive services for alterations of the mouse genome and for storage and distribution of genetically altered mouse lines. These services include direct targeted alterations of the mouse genome via CRISPR/Cas9 targeted DNA cleavage/repair (non-homologous and homologous) as well as standard DNA transgenics, introduction of ES cells into blastocysts, and an array of support services focused on safe storage, rederivation, and sharing of mouse models. The full array of services is listed below.
Consultation services. The GAMC Director and Technical Director provide consultations free of charge regarding animal husbandry, microsurgery, oversight regulatory guidelines, and use of specific mouse strains.
- Direct Genomic Modification by Targeted Endonucleases. This service relies most heavily on RNA directed nuclease activity (CRISPR/Cas9). Injections of CRISPR/Cas components (RNAs and proteins) can be nuclear or cytoplasmic depending on specific reagents and goals. For a subset of projects, the targeting reagents can be introduced by electroporation rather than by microinjection, yielding substantial savings in time and effort. Template double strand DNA can be co-injected with the CRISPR/Cas reagents to generate high-fidelity homologous recombination. These GAMC functions are closely coordinated with those of the separate Perelman School of Medicine (PSOM) CRISPR/Cas9 Mouse Targeting Core, directed by Dr. Jorge Henao-Mejia (Immunobiology) where investigators can receive expert advice on best approaches to direct genomic modifications and have all necessary reagents generated for them prior to injection by the GAMC. Although the highly specialized ‘tool sets’ of the GAMC and CRISPR cores are entirely distinct, they work in close communication to maximize the efficacy and success of corresponding approaches to the generation of mouse models.
- Standard pronuclear microinjection of DNA (sizes extending up to P1, BAC, and YAC) into mouse zygotes. This service is paired with genotyping of tail DNA of live-born pups to identify positive founders. "Transient transgenic" services with generation of pregnant females at defined gestational ages also is available.
- Blastocyst microinjections. Mouse ES cells with defined genomic modifications are transferred to the TCMF for microinjection into blastocysts.
- Embryo and sperm cryopreservation. Standard approaches to cryopreservation are in use as well as a high-throughput procedure in which two-cell embryos generated by IVF are cryopreserved.
- In vitro fertilization (IVF). This technique is useful for maintenance of lines with impaired fertility due to a variety of issues and in conjunction with sperm freezing. The zona can be laser ‘conditioned’ when necessary to increase efficiency of sperm penetration.
- Oocyte harvest service. The GAMC provides users with one-cell embryos for pronuclear microinjection.
- Special strain requests. This service is beneficial to investigators whose studies are dependent on the use of mice with inbred genetic backgrounds.
- Embryo rederivation. Embryo re-derivation is of particular use in expediting transfer of the colonies between certain vivaria and to shorten quarantine times of imported mouse lines.
- Intracytoplasmic sperm injection (ICSI). This piezo drill technique is primarily used to increase the fertility of cryo-preserved sperm that demonstrate defective motility/egg penetration.
- Training. Users of the GAMC are trained in aspects of mouse husbandry and microsurgery that must be implemented outside of the GAMC to facilitate services such as cryopreservation, embryo harvests, and line rederivations.
- Web-accessible protocols and information. The GAMC maintains an extensive set of protocols on its web site for preparing DNAs and ES cells for microinjection as well as tips on optimizing animal husbandry.
- Collaboration with other Shared Resources and Institutional cores. Close integration with the ACC Genomic Analysis Core is used to genotype founders generated from routine transgenic services. The GAMC works in close cooperation with CRISPR/Cas9 Mouse Targeting Core — the majority of genome modification projects currently originate in the CRISPR core.
Technologies and Major Equipment
The 487sq ft GAMC laboratory is housed in two adjacent HEPA-filtered rooms in the Clinical Research Building vivaria. This laboratory contains four fully equipped microinjection stations, one equipped with a piezo drill and a second dedicated for laser procedures. Other equipment includes: four stereomicroscopes, two Pipette pullers and microforges, a water bath, pH meter, two iMACs, two Eppendorf microfuges, refrigerator, freezer and liquid N2 dewar, a Nikon Coolpix camera and a color monitor, Biocool III apparatus, two CO2 incubators, two dry sterilizers, an XYClone Laser System, and a BTX electroporator ECM 830.
For more information, visit the Facility's website: https://www.med.upenn.edu/genetics/tcmf