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Gene targeting

Vivarium and Transgenics Core

Service overview

Gene-targeted mice, called knock-out or knock-in mice, are produced by injecting blastocysts with embryonic stem (ES) cells that have been genetically modified. ES cells are pluripotent stem cells that are derived from the inner cell mass of 3.5 days post coitum (DPC) embryos called blastocysts. These undifferentiated cells have the capacity to differentiate into most mouse tissues. 

Typically, 129/SvJ strain ES cells are transfected by electroporation with an isogenic DNA construct containing homology to a region of the genome. Through homologous recombination, this DNA fragment is inserted into the homologous gene either “knocking out” the function of the gene or “knocking in” a functional gene into the same locus. 

The transfected cells are placed into selective media and the surviving clones are isolated, grown in culture, replicated, and placed in frozen storage. The cells of one replicate are processed for DNA isolation and clones testing positive for homologous recombination are thawed and expanded. These positive clones are injected into blastocysts from a donor mouse and implanted into pseudopregnant foster mothers. Chimeric offspring are backcrossed to test for germline transmission of the targeted gene. Heterozygous mice are then intercrossed to derive homozygous gene-targeted mice.

Contact Audra Guikema with questions and for pricing on any of the services listed below. Please request services via iLab. New users can sign up by visiting iLab here and clicking the “Sign up” link in the top right corner of the page.

Specific services include:

Electroporation of ES cells, clone selection, and DNA isolation

Electroporation will be scheduled upon receipt of the linear DNA from the user. Samples are prioritized by the date received.  Users will be notified of the electroporation date and the expected date for DNA sample receipt. Please see this protocol for specific preparation requirements.

During the procedure, DNA is electroporated into ES cells (CJ7, R1, etc.)  After electroporation and selection, several hundred clones are isolated, cultured in multi-well plates, and replicated. One replicate plate is placed in frozen storage and DNA is isolated from each clone in the other replicate. 

The DNA samples are then shipped on dry ice to the user for genotype analysis. Generally, DNA is delivered to the user three weeks from the electroporation date. Users are responsible for paying the shipping charges for the DNA samples. 

Since storage of frozen cells in multi-well dishes is not optimal, it is strongly suggested that users return the genotype results to the Vivarium and Transgenics Core within four weeks of receipt of the DNA samples.

Thawing and expansion of ES cell clones

After the user has identified the positive ES cell clones by Southern blot analysis or PCR, five clones are thawed and expanded. More clones can be thawed at additional expense. Cryopreserved ES cell stocks are then prepared from each positive clone and double-banked. DNA also is isolated from each positive clone for secondary confirmation or archival storage by the user. VARI will ship the DNA samples or ES cell frozen stocks to the user by FedEx at the user’s expense.