RRC-TPS: Gene Targeting in ES Cells
Gene targeting vectors (review our protocol for preparing DNA for electroporation) submitted to the TPS will be electroporated into germline-competent ES cells. To achieve efficient homologous recombination in ES cells the use of isogenic DNA to prepare your targeting vectors is strongly encouraged. Contact the TPS Director to discuss the appropriate DNA source before you construct your vector. Assistance with targeting vector design is also available.
Following electroporation, cells will be grown in drug-selection medium, after which individual drug-resistant colonies will be picked into 96-well plates (three to four plates). Colonies grown in the 96-well plates will be split further into four 96-well plates, two of which will be frozen at -80oC for future use and two used to prepare DNA. The two plates containing DNA will be provided to the investigator for genotyping (colony screening) to identify homologous recombinants (targeted clones).
Potentially targeted clones (up to 4) identified by the investigator will be thawed from the frozen 96-well plates and expanded, and the cells will be frozen in cryovials for long-term storage in LN2. At the same time, cell pellets will be provided to the investigator for DNA isolation and another round of genotyping to confirm gene targeting. Once targeting is confirmed, metaphase spreads will be prepared from the appropriate clones to determine that the cells are euploid. The clones will also be tested for mycoplasma contamination. Suitable clones will be prepared for ES cell microinjection into embryos.
Preferably, genotyping is done by Southern analysis, which requires 5’ and 3’ probes located outside the regions of homology and restriction fragment polymorphisms between the targeted and wild-type alleles. A unique, or diagnostic, restriction site can be introduced with the positive selection cassette (neo) if necessary. Each probe must be able to clearly discriminate between the two alleles. Ideally, the targeted allele will produce a hybridization band that is smaller than the band generated by the wild-type allele; this eliminates potential false positives caused by incomplete restriction digestion.
Before constructing the vector, develop an assay for your 5’ and 3’ external probes. Using isogenic DNA, confirm that each probe will detect a single wild-type band in 2-5 ug DNA. If you are using ES cells from the TPS, the facility can provide you with a cell pellet from which you can isolate isogenic DNA to develop your assay.
Ultimately, genomic DNA from drug-selected colonies grown in individual wells of 96-well plates will have to be screened. Once you have worked out your screening assay we will provide free practice 96-well plates that you may use to perfect restriction enzyme digestion and Southern analysis. A protocol will also be provided.
PCR may also be included in your screening strategy. Note, the PCR screen will amplify a novel junction fragment created by homologous recombination; it will not amplify either the wild-type allele or a random integrant. PCR screening to identify homologous recombinants is often performed using one primer from the positive selection marker and a second primer from endogenous sequences just outside one arm of homology. The assay generally requires that the selection marker be inserted at an asymmetric location near one end of the homologous sequences, resulting in a “short arm” of homology and a “long arm” at the other end. While PCR screening is much quicker than Southerns, using a shorter arm may reduce the frequency of homologous recombination.
In order to test the PCR conditions and to generate a positive control for your PCR screen, a control vector should be constructed, and you must demonstrate sensitivity down to the single-copy level. This vector will include the two primer annealing sites, and can be made by cloning a small piece of genomic DNA just outside the short arm of homology. Although the vector can easily be designed to generate the exact product expected from the targeting event, it is preferable to design the vector in such a way as to generate a PCR product that is different in size from the product created by the targeted allele. To work out your PCR conditions, mix 1 pg control plasmid for every 1Kb of its length with 5 ug isogenic DNA. Once you have optimized your PCR assay we will provide free practice 96-well plates that can be used to simulate the colony screen. A protocol will also be provided. These PCR reactions will contain an average 0.2 ug genomic DNA and should be mixed with 0.04 pg control plasmid for every 1 Kb of its length.
While you are designing your screening strategy, you should also determine the method that you will use to confirm removal of the selection cassette by recombination at lox (or Frt) sites; and, for conditional alleles, removal of your target exon(s) after recombination at lox (or Frt).