Molecular Genetics Service Core

a. Production of Transgenic Mice: Transgenic animals are produced by injecting a DNA construct directly into the pronucleus of a newly fertilized mouse embryo or by electroporarted target DNA into one-cell embryos. Plasmid or BAC constructs can be used for microinjection. We use C57BL/6J inbred mice for these manipulations, however; we can accommodate other strains (e.g. F1 hybrids) by special request. 

We charge per injection/electroporation day and we typically do 2-4 injections.  The pups are screened for the presence of the transgene by a polymerase chain reaction genotyping assay. When the founder animals are at weaning age they are transferred to the investigator.   Transgenic founders are normally received seven to eight weeks after the original injections are performed when the mice are four to five weeks old.  

b.CRISPR/Cas-mediated Genomic Engineering: The use of embryonic stem (ES) cells has been the traditional method to produce gene targeting in mice, which is costly and time consuming, generally taking 9-12 months to generate single gene targeted offspring. This technology has been superseded by CRISPER/Cas-mediated genomic engineering where mice can be produced carrying multiple mutations, endogenous reporters, or conditional alleles. In this technology, specifically designed nucleotides are injected or electroporated directly into the one-cell embryo circumventing the need for selection of ES cells and chimera formation. Following target design, the generation of gene targeted mice cane be achieved in as little as 4 weeks. The molecular Genetics core has implemented all phases of this technology and is available to discuss individual needs. See Cell 157:1262, 2014 and Nature Protocols 9:1956, 2014 for more information.

c. Production of Chimeric Mice with ES Cells: The MG core maintains the option for microinjection of validated ES cells. This service aims to assist those projects where CRISPR/Cas9 modification is not optimal, such as creating a conditional allele containing two loxP sites flanking a designated exon. However, ES cell targeting, screening, clonal expansion and validation are the responsibility of individual labs. Targeted ES cells for many mouse genes are available from KOMP/EUCOMM, allowing the production of conditional (floxed) alleles. The ES cells ordered from KOMP/EUCOMM should be karyotyped and sequence validated before microinjection (https://www.mmrrc.org/catalog/general_ES_cellline_fee_resequencing.php). Also, a few custom ES cell gene targeting services are available from other sources, such as AMC at UNC https://www.med.unc.edu/amc/services/gene-targeting-services/.

We will generate chimeric mice by injecting the expanded (validated) ES cells into C57BL/6J blastocysts in our core animal procedure room. The resulting chimeric mice will be transferred to the investigator’s mouse facility 7-8 weeks after the original blastocyst injections. We charge the microinjection service per injection day, and we typically do 4 injections.

Dr. Chen will meet one-on-one with our investigators to explain the CRISPR/Cas technology and alternative approaches, and discuss how it might best be incorporated into their research strategies.

Freezing mouse embryos or sperm is a convenient way to preserve important mouse lines for possible future use while saving animal care per diems. Receiving mouse strains of interest from other institutions can be held in the LN2 freezer for subsequent resuscitation of the line by IVF.

-Sperm Cryopreservation is the preferred method for long term storage of mouse strains. The cost to freeze a line is minimal. Typically the method requires that 2 males of the strain of interest are sacrificed to harvest sperm for freezing. The cost is for each strain. Sperm will be maintained in liquid nitrogen and an annual storage fee will be assessed. 2 males, 10-20 weeks of age that are healthy and reproductively active are typically enough to guarantee recovery. Males are brought to 710C McMillan for sperm freezing.

-Embryo Cryopreservation is an alternative way of long-term preservation of mouse strains. Typically, seven to ten young male mice with your gene of interest will be needed to generate enough embryos for each freezing session. The males are transferred to SRF-W animal room 111B for mating. While the males are in room 111B animal care per diems will be billed directly to the PI. Wild type females, 3 weeks of age will be purchase by the PI and sent to room 111B. The delivery dates will be designated in a pre-planned schedule. The females are sacrificed and the embryos harvested and frozen down at the 8 cell stage. It may take several freezing sessions to freeze down enough embryos in the bank. The cost is for each session. A test thaw to blastocyst stage is done each session for each strain. Frozen embryos may also be send from other institutions for recovery in our facility. The fee is for the females used as recipients for the embryos. Frozen embryos will be maintained in liquid nitrogen and an annual storage fee will be assessed.

Fresh sperm from 2 males or frozen sperm samples can be used for IVF. 20 wild type females are purchased and used for oocyte donors.

Consultation and training in PCR techniques and breeding strategies.

Mice that have been exposed to pathogens or transfers of “dirty” mice from other facilities can be made free of disease through the rederivation. The Division of Comparative Medicine (DCM) currently provides rederivation services for the infected mice. If you plan to import dirty mice from another institution, please consult Susan Penrose for the options and details about DCM rederivation services.