Gene Targeting Service Animal Genomics Services


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The Gene Targeting Service (GTS) creates genetically engineered (“knock-out”) mice via homologous recombination in embryonic stem (ES) cells. Our services can be ordered together or individually depending on the investigator's needs.

Gene targeting in mice involves several important steps:

Engineering a gene targeting construct.
Transfection of embryonic stem (ES) cells.
Assessing ES cells for homologous recombination.
Generating chimeric mice by blastocyst injection.
Assessing germline transmission.
Breeding chimeric mice to homozygosity.

Each of these steps requires close coordination between the investigator and the GTS. Please read the following guide before submitting your gene targeting request. If you have conditions or requests that differ from these, please contact us to make revised or additional arrangements.

Getting started

We recommend strongly that you discuss your needs with the GTS before beginning a gene targeting protocol. We can help you design your construct (if necessary), tell you more about our expectations for your role in the gene targeting procedure, and clarify any issues pertaining to fees. We also can discuss strategies for conditional and tissue-specific mutagenesis, and can provide maps and plasmids containing various elements necessary for different targeting strategies.

When you are ready to speak with us, please submit the on-line form, and we will contact you to arrange a meeting.

Engineering a gene targeting construct

Based upon our observations with successful targeting constructs, we urge you to design your construct to incorporate at least the following characteristics:

use DNA from mouse strain 129/Sv to optimize recombination efficiency
provide at least 5 kb of total flanking homology (minimum 1 kb for either targeting arm)
provide a +/– selection system (e.g. neo/Tk)
have the neo gene (conferring resistance to G418) driven by a ubiquitous promoter, such as PGK or PMC

After the construct is engineered we will review your construct data with you to confirm that the construct design and DNA concentration are appropriate for transfection of ES cells. We will help you correct any potential problems before proceeding with transfection. We also will discuss your strategy for screening individual clones for correct homologous recombination to verify that the necessary elements are present or in progress. We also can provide you with 129Sv genomic DNA from the appropriate ES cells to confirm that your hybridization protocols (Southern blots, PCR, etc) can detect the desired gene targeting event.

Transfection, selection and assessment
of embryonic stem (ES) cells

ES cells used by the GTS have a verified karyotype (40 chromosomes) and are free of adventitious infectious agents such as mycoplasmas. Your construct will be transfected into ES cells by electroporation. Following successful electroporation, selection and clonal expansion, we will provide you with approximately 200 clones (unless a different number is required) on 24-well plates from which you will extract DNA and screen for homologous recombination. We freeze parallel 24-well plates at –70C, so screening must be performed promptly. Individual clones demonstrating homologous recombination events will be thawed at your request and you will receive a second dish of cells to confirm the initial screening assessment.

Karyotyping

At the time of thawing and expansion of positive clones, we recommend that you have the cells karyotyped, due to the frequency of nondisjunction events in ES cell cultures and failure of clones with more than 40 chromosomes to generate healthy, transmitting chimeras. We perform karyotyping simultaneously with freezing stocks of each clone so results are known prior to blast injection.

Blastocyst injection, generation of chimeric mice

C57Bl/6J blastocysts will be injected with positive clones and transferred to pseudopregnant CD-1 females. Recipient mice will be transferred to your census sheet at this time. Highly chimeric progeny (assessed by coat color) that result will be mated with mice of the appropriate coat color to assess germ line transmission of the targeted ES cells. Once germ line transmission is achieved, mice will be transferred to the users’ animal room. Access the animal relocation request form online. Subsequent management of the founder line can be requested through the YARC Rodent Service, if desired.

Conditional mutagenesis

We can work with you to generate conditional targeted mutants. We have vectors and mice available to enable use of Cre/loxP and Flp/Frt recombination to generate the desired mutants. Contact us as you would for "standard" gene targeting experiments, and we can plan the additional arrangements. Please note: certain technologies, such as use of Cre/loxP, are covered by patents and license agreements. Use for academic research is generally approved, but applications designed for “profitable”uses should be cleared with the Office of Cooperative Research.

Gene Targeting Service Order Form

Blastocyst Injection Order Form