ESC-mediated Gene Targeting
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
Submitting Your Request
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 and resources available for conditional and inducible mutagenesis, and can provide maps and plasmids containing various elements necessary for different targeting strategies. It helps us to be aware of any unorthodoxstrategies you plan to use, especially when they improve efficiency of the process! In addition, we can discuss the optimal combination of ES cells and blastocyst embryos for your project.
When you are ready to speak with us, please submit the on-line GTS Service 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 isogenic to the ES cell line to be used to optimize recombination efficiency. We typically recommend 129/Sv or C57B1/6, but other ES cell strain backgrounds are available.
- provide at least 5 kb of total flanking homology (minimum 1 kb for either targeting arm; recommended minimum 2kb)
- provide a +/– selection system (e.g. neo/Tk, neo/DT)
- use a ubiquitous promoter, such as PGK or PMC, to drive the neo gene (conferring resistance to G418)
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 ES cells from which to extract genomic DNA to confirm that your screening protocols (Southern blots, PCR, etc) can detect the desired gene targeting event.
Targeting Vector Construction
On a limited basis, we are now offering vector construction as a fee-for-service. We will meet with you to discuss the project and design the construct to your specifications, and carry out the necessary vector production. Please call us for details, fees, and availability.
DNA Submission
Gene targeting constructs (50 µg minimum) should be linearized with an appropriate restriction enzyme, extracted, precipitated, and resuspended at 1 µg/µl in TE or similar buffer.
Scheduling
Constructs will be electroporated in the order they are submitted. The appropriate on-line GTS Service form, including charging information, shall be sent prior to or at the time of submission. Non-Yale investigators may email with PO billing arrangements or call for credit card purchasing.
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
Unless another strain is specified, C57Bl/6J blastocysts will be injected with positive ES 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. If C57Bl/6-derived ES cells are injected into C57Bl/6 blastocysts, coat color cannot be used to determine chimerism. In this case, we will provide you with tail biopsies for genotyping, and positive chimeras will be mated. Once germ line transmission is achieved, mice will be transferred to the users’ animal room. Subsequent management of the founder line can be requested through the YARC Rodent Service, if desired.
Injection of Cells from Outside Sources
We will be happy to inject targeted ES cells generated in your lab or obtained from an outside source such as KOMP or EUCOMM. Such ES clones are subject to mycoplasma testing for a small fee. We cannot, however, guarantee anything about the performance of such cells in generating chimeras or germline transmission, although we are happy to report that we have a high rate of success using lines from the various large-scale gene-trap and knockout projects.
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.
Animal Use Approval
Investigators must have an IACUC-approved protocol for use of mice prior to initiation of the experiment.
Success of Experiments
Due to inherent variability in gene targeting experiments, and the variety of constructs submitted, GEC cannot guarantee the success of any particular experiment. However, we will guarantee that we will make every effort possible, through consultations and troubleshooting, repeats and redesign if necessary, to work with you to achieve correct gene targeting and germline transmission.