Design & Generation of gene targeting constructs
Ideally gene targeting vectors should be based on genomic DNA that is isogenic to the mouse ES cell line that will be used in experiments to ensure high targeting frequency. E19Tg2aIV (129P2/OlaHsd) and JM8 (C57BL/6N) are currently available.
After the isolation of genomic clones for the gene of interest and generation of a detailed restriction map, the targeting vector should be designed. Genomic regions that can serve as Southern blot probes to recognize targeting events should be tested on Southern blots of isogenic genomic DNA from ES cells. This will ensure future rapid genotyping of both ES cells and mice. Alternatively, a well design long range PCR can be used in combination with Southern blot.
It is imperative that the methodology for detecting recombinant alleles has been worked out prior the initiation of experiments. Wild type ES cells in 96-well format, as well as purified genomic DNA, can be provided as necessary.
Please be sure to develop a good genomic map of the targeted locus and unique probes that are external to the targeting construct BEFORE beginning gene targeting experiments. NB-After extraction of the DNA from drug-resistant ES clones, there is only enough DNA for a single restriction digest and Southern analysis. Note that the plates of frozen replica plates with ES cells cannot be stored longer than 1 month. Delaying identification of targeted clones beyond that time runs the risk of the loss of the clones. Re-targeting of ES cells will be recharged at full cost.
Researchers must provide evidence of a successful strategy to identify positive homologous recombination events by both Southern blot analysis and PCR. This evidence shall consist of construct maps as well as PCR and Southern blot results showing the expected endogenous bands.
Probe sequences used to identify homologous recombination events must be located outside the genomic sequences used to create the targeting vector. These probes, when hybridized to a Southern blot of restriction enzyme digested 129/SvJ or C57BL/6N genomic DNA should identify a single copy of the targeted gene (the endogenous gene).
Once the construct is complete and genotyping strategies approved, the client will submit a minimum of 40 µg of linearized, purified vector DNA at a concentration of 1 µg/µl in sterile water. Alternatively, the GTF can linearize and purify the targeting DNA construct for the customer, see price list.
Upon receipt of the linear DNA from the customer, the electroporation will be scheduled. Samples will be prioritized by the date received.
Customers will be notified of the electroporation date and the expected date for their receipt of ES clones or DNA samples.
DNA will be electroporated into preferred background ES cells (currently 129Sv or C57BL/6N ES cells)
After electroporation and selection, clones will be isolated, cultured in multi-well plates, and replicated.
One replicate plate will be placed in frozen storage and the other replicate plate will be delivered to the customer.
The DNA samples will be transferred / shipped on dry ice to the customer for genotype analysis. Generally, the DNA will be delivered to the customer three weeks from the electroporation date.
Customers are responsible for covering all charges associated to delivery of the DNA samples.
On request GTF can genotype positive ES cell clones by long range PCR and only PCR positive clones will be shipped to the customer. This service will entail additional costs.