Transgenic/Knockout Rodent Core Facility
The Transgenic/Knockout Core was established in 1993 to produce transgenic and knockout mice for USC Norris Cancer Center investigators and to increase the use of mouse technology through education and consultation to the cancer research community. Since its inception, the Core has operated under the faculty leadership of Robert Maxson, Ph.D. and manager, Nancy Wu, M.D. and is widely recognized within the USC community and beyond for the high quality of its services. The Core produces transgenic and knockout mice and now offers two important new services, knockout rats and CRISPER/Cas9-mediated gene editing in mice. Ancillary services include vitro fertilization, re-derivation of mouse strains, and cryopreservation of embryos with the overall goal to offer state-of-the art services for the manipulation of mouse and rat genomes as part of the Cancer Center Support Grant (CCSG). The facility is located centrally on USC’s Health Sciences Campus.
Please contact Nancy Wu (Core manager) at email@example.com or 323-865-0726 to discuss the rates and schedule for services. (Please remember to get your IACUC approval to start).
Order Forms are available online for the following
Remember that all work needs both Institutional Animal Care and Use Committee (IACUC)
and USC Institutional Biosafety Committee (IBC)
Major Services, Technologies, Equipment and Expertise Provided
- Knock-Out Rats:
Using ES cell technology pioneered by Qilong Ying, PhD at USC, the Core has generated the first knockout rats using a protocol developed by Dr. Qilong Ying of the Broad Center for Regenerative Medicine and Stem Cell Research (Li et al, Cell, 2008). As such, the Core has added ES cell injection and gene knockouts in rats to its complement of services.
- CRISPR/Cas 9 Technology:
The new CRISPR/Cas9 technology will make it possible to produce gene knockouts much faster and at lower cost than current ES cell-based technology. Based on a bacterial immunity system, the type II bacterial clustered, regularly interspaced, palindromic repeats CRISPR-associated (Cas) system has shown itself to be a powerful tool for editing genomes (Yang et al, JRNL, 2013). This technology makes the process of generating knockout animals much easier and cheaper (approximately the same time and fee as a transgenic mouse) than standard ES cell-based technology.
Technology and Major Equipment
- Consultation: The Core offers expert consultation on vector preparation, developmental biology, and approaches to testing gene function in rats and mice. Dr. Maxson advises PIs on research strategies and Dr. Wu meets with research staff to discuss their orders.
- Direct Services:
- Production of Transgenic Mice: The Core offers expert consultation on vector preparation, developmental biology, and approaches to testing gene function in rats and mice. Dr. Maxson advises PIs on research strategies and Dr. Wu meets with research staff to discuss their orders.
- BAC Transgenics: The Core offers BAC injection, which entails injecting large (up to several hundred KB) DNA fragments into zygotic nuclei.
- Production of Gene Targeted Mice/Injection of ES cells into blastocysts: Clones (usually two) that have been demonstrated to have normal karyotypes are injected into blastocysts. If clones are from strain 129Sv ES cells, then they are injected into blastocysts from C57Bl/6 mice. If they are from C57B/6, then they are injected into C57BL/6J-Tyrc-2J /J mice. Pups are assessed for coat color chimerism. Those found to be highly chimeric are returned to users for breeding to obtain germ line chimeras.
- Production of Gene Knockout Rats: This service involves the injection of targeted ES cells into rat blastocysts, implantation of the injected blastocysts into pseudopregnant rats, and maintenance of pups up to three weeks in the facility.
- Genome Editing by means of CRISPR Technology: Type II bacterial clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system is a powerful tool for editing genomes. The system consists of the Cas9 nuclease and a single guide RNA (sgRNA). The guide RNA, 20 nucleotides in length, guides the Cas9 nuclease to the target sequence, where it introduces double stranded breaks. These are repaired imperfectly by the process of non-homologous end joining. The sgRNA together with Cas9 (RNA or protein,) are injected into a zygote. Alternatively, Cas9 is supplied by the zygote as the product of a transgene. Coinjection of a DNA fragment with homology to the sequences flanking the double stranded break can produce mutant alleles with inserts such as loxP sites. The Core injects the Cas9 protein and sgRNA into single-cell zygotes, and implants the zygotes into recipient females.
- In Vitro Fertilization: Mouse strains are sometimes preserved as frozen sperm. Thus it is important to have the ability to regenerate such strains by in vitro fertilization. Females are superovulated by hormone injection, and unfertilized eggs are collected and combined with sperm, which can be fresh or frozen, in a special buffer. Fertilized eggs are incubated overnight and several 2-cell embryos are implanted in pseudopregnant recipient females.
- Rederivation of Transgenic and knockout Lines: Investigators often wish to import mice that carry pathogens or have been kept in contaminated facilities. USC policies do not permit such mice to be maintained in USC Department of Animal Resources. The Core sets up matings, removes fertilized eggs and transfers them into clean pseudopregnant foster mothers.
- Cryopreservation of Blastocysts: The high cost of maintaining a strain that may be important for long-term research objectives, but is not being used at the present time, sometimes forces the investigator to eliminate mice that would otherwise be maintained. The Core obtains 100-300 blastocysts from the strain to be preserved, freezes such blastocysts according to methods in use at the Jackson Laboratory, and stores them in liquid nitrogen. Strains that the Core has successfully preserved include C57BL/6J, 129, B6D2F1, as well as mixed strains.
Education. The Core is committed to educating and encouraging cancer investigators to use mouse and rat technology in their basic/translational research. It serves as a source of information about mouse husbandry, genotyping, genetics, and histopathology, providing advice to a large number of investigators.
- Microinjection Facility: The microinjection room consists of a 196 sq. ft. laboratory in the ZNI vivarium with two microinjection stations. Both are equipped with Leica DMIRE2 inverted research microscopes and Eppendoff micromanipulators. A Wild and a Nikon dissecting scope are available for surgical procedures such as transferring and implanting eggs. The Core is also has a Sutter Instrument Flaming/Brown Micropipette Puller.
- Cell Culture Facility: The cell culture facility consists of a 400 sq. ft. room in the ZNI facility equipped with two cell culture hoods a Nikon inverted microscope, and two desk top centrifuges. In addition, a liquid nitrogen freezer is available for storage of cells.
- Mouse Holding Rooms: The Core includes one room of dedicated vivarium space in the ZNI vivarium. The rooms are pathogen-free and stringent measures are taken to maintain them as such. The Facility now houses approximately 500 cages of mice. The total capacity of the ZNI vivarium is 7,000 cages.
All Core personnel have extensive experience in mouse and rat knockouts and transgenics. Each has been with the core for many years, which has contributed greatly to the high success rate and overall satisfaction of its user base. Robert Maxson, PhD, founded the Core in 1993 and has served as the Facility Director since then. Dr. Maxson was trained at UC Berkeley and Stanford University. A well-established mouse geneticist and developmental biologist, he has assisted many Cancer Center investigators in developing mouse models. He continues to be the thought-leader of the Core, bringing advances in the field to the Core and providing expert scientific advice to Cancer Center members. Nancy Wu, MD, Research Scientist has also been a part of the Core since its founding and is an expert in the production of transgenic and knockout mice and rats. Youzhen Yan, MD, trained in microinjection procedures, assists Dr. Wu.