Core Director: Pamela L. Mellon, PhD
Professor of Reproductive Medicine and Neurosciences
The DRC Transgenic, CRISPR Mutagenesis, and Knock-out Mouse Core is a state-of-the-art facility that has an outstanding track record in the production of genetically altered mice. Transgenic mice carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs.Mice carrying directed mutations are created by injection of CRISPR guide RNAs with Cas9 mRNA/protein and template DNA. Knock-out mice lacking specific genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric mice. Highly experienced personnel produce transgenic, CRISPR, and knock-out mice for DRC investigators at very reasonable cost and with very short lead times. The Core provides embryonic stem cell recombination, knockout mice, CRISPR mutant mice, transgenic mice (both standard and BAC transgenics), sperm or embryo cryopreservation, and pathogen-free embryonic rederivation to the DRC community at discounted rates. This UCSD-based Core Facility has been in operation since 1992.
Transgenic Core Request For Services This form MUST be filled out before beginning any work with the Core.
A desired DNA construct is injected into the pronucleus of a 0.5 d.p.c. embryo. Resulting pups are returned to the client. The client will then analyze these by PCR or Southern blot to determine the presence of the given transgene.
CRISPR Mutagenesis and Internal Deletion
For internal deletion, a CRISPR guide RNA that targets the desired gene region is coinjected with commercial Cas9 mRNA or protein. For specific mutations or insertion/deletions, a CRISPR guide RNA that targets the desired gene region is coinjected with commercial Cas9 mRNA or protein and a template DNA that harbors the mutation. Generally, these are injected into the cytoplasm of fertilized one-cell embryos. Resulting pups are returned to the client. The client will then analyze these by PCR and sequencing to determine the presence of the given transgene.
Mouse embryonic stem cells that have undergone homologous recombination are injected into the blastocoel cavity of 3.5 d.p.c. blastocyst stage C57Bl6 (black or albino as needed) embryos. Injected blastocysts are implanted into pseudopregnant recipient females, and chimeric pups are born. Chimerics are returned to the client. Those that transmit the 'knock-out' allele to germline produce agouti pups, which are analyzed for phenotype by the client.
A targeting vector containing a 'knock-out' or 'knock-in' gene of interest is electroporated into mouse embryonic stem cells (C57Bl6). The cells undergo drug selection, clones are picked and expanded in tissue culture, then returned to the client for analysis. Homologous Recombinant clones are verified by the client using either PCR or Southern Blot analysis by the client.
Chromosome counting is done on targeted homologous recombinant ES cell clones prior to blastocyst injection for quality control.
Embryo rederivation is used to create a pathogen-free strain of mice from lines that arrive at the University from a non-approved vendor and mice contaminated with various style="margin-top: 5px; margin-bottom: 15px; margin-left: 20px;"pathogens (MHV, Pinworm, Parvovirus). Males arrive in the Rodent Isolation Unit (RIU), and wild-type females of the desired background strain are superovulated and mated to the males. Embryos are harvested in the RIU, washed and transferred into pathogen free, pseudopregnant recipient females, which are returned to the client.
Sexually mature females that are infertile or cannot carry a litter to term are ovariectomized. These ovaries are then transplanted into the wild-type ovariectomized females of a given background strain, and immediately returned to the client. Two weeks later, the recipient females are mated by the client to produce transgenic pups of the original genotype.
Embryo or Sperm Cryopreservation
Genetic banking through the use of mouse embryo and sperm cryopreservation is a practical means to store scientifically valuable mice. Mouse embryos or sperm stored in liquid nitrogen offer a safe way to preserve lines with potential future use but no current use and offer savings in facility space and the expense associated with keeping a breeding colony. Another advantage is that during the process of embryo or sperm collection, most pathogenic organisms are excluded as in other methods used to rederive lines of mice.
Briefly, the embryo cryopreservation procedure entails harvesting embryos from donor females at the 8-cell developmental stage that is optimal for cryopreservation by our method. A controlled rate methanol bath freezer is used to gradually cool the embryos to -80°C, before plunging them into liquid nitrogen for storage. Quality control is assured by freezing and thawing a representative sample of wild-type frozen embryos along with each line frozen down. Frozen embryos should be retrieved by the investigator one week post-freezing for long-term storage in liquid nitrogen in the investigator's own lab.
Alternatively, sperm can be cryopreserved. In this case, at least two proven male breeders between the ages of 3-5 months are provided to the Core. Core cryopreserves sperm and stores in LN2 for the client. Client does long-term storage in LN2.
our detailed website for our current rates. The DRC rates are the same as those shown for Cancer Center Members.
|Transgenic, CRISPR Mutagenesis, and Knock-Out Mouse Core|
|Pamella Mellon, PhD|
Professor of Reproductive Medicine and Neurosciences
Transgenic Mice, CRISPR Mice, and Cryopreservation
Gene Targeting, Embryonic Stem Cells, and Blastocyst Injection