What about knockins, targeted point mutations and conditional knockouts?
     We do those too. The technology is the same, although construction of the targeting vector might be a little more involved. We have done numerous knockins, targeted point mutations and conditional knockouts. Come discuss strategy with us, and we'll put you on the right track. (the cost is the same)
     For all gene targeting, you must be able to verify that targeting has occurred correctly by Southern blot analysis with probes external to the two ends of the targeting vector, but this is particularly important for knockins, targeted point mutations and conditional alleles. In addition, verification that the targeting vector is exactly as you expect should be verified by sequencing of critical regions, such as coding sequences in knockins, and loxP sites in conditional alleles. For conditional alleles, we would like you to demonstrate that all the loxP sites are functional. We provide detailed information about the design of gene targeting vectors.

What can be done to ensure success?
     Typically, one wants to ensure that no protein is made from the targeted gene. Therefore, construct a vector to delete as much coding sequence and as many functional parts of the protein as possible. However, there is a practical limit to the amount of DNA that can be deleted by targeting: gene targeting frequency decreases with deletions greater than 10 kb.
     Include as much homologous DNA as possible: the greater the amount of homologous DNA, the greater the possibility that targeting will have a reasonable frequency. At a minimum, no arm should be less than 1 kb and the two arms together should total more than 7 kb. The more homologous DNA you use, the better.
     Construct your vector with genomic DNA from the 129 strain of mice to target an ES cell line from the 129 strain of mice. Small differences in DNA sequence can have very large effects on targeting frequency. 129 genomic clones in BAC vectors (.pdf) can be purchased from the Sanger Institute. BACs containing your gene can be identified using the ensembl genome browser, selecting the DAS source "129S7/AB2.2 clones". If you wish to obtain a specific clone, clicking on the BAC will bring up a menu, and selecting the link at the bottom of the list will take you to the order form.
     Alternatively, DNA from C57BL/6J BAC genomic clones can be used to construct targeting vectors if there are no polymorphisms in the region of the gene between C57BL/6J and 129S1/SvImJ strains of mice. The locations of single nucleotide polymorphisms can be ascertained here on the Jax web site, and C57BL6J BAC clones can be identified with the UC Santa Cruz genome browser and purchased from BACPAC Resources Center CHORI.
     Although we know many of the parameters that affect gene targeting frequency, we clearly do not understand all. Targeting vectors with small differences (e.g. the pieces selected for the arms of homology or the orientation of the selection cassette) targeted to the same gene can have very different targeting frequencies. Moreover, it can be difficult and time-consuming to build constructs for gene targeting. Given the uncertainty of whether targeting will occur at all, think twice before investing a lot of time in building a complex, kitchen-sink vector. It would be better to invest effort in designing multiple simple vectors.
     Additional information about the design of targeting vectors has been excerpted and modified from LePage and Conlon, Methods Mol Med 2006.

How will I know if it worked?
     In order to verify if gene targeting occurred, and in order for us to begin the gene targeting process, you must have developed a proven strategy to determine if gene targeting occurred. You will need a probe that is not contained in the targeting vector that will distinguish the targeted from the wild type allele. Do not use enzymes that contain CpG's as these are frequently methylated in ES cell DNA.
     We require that you have demonstrated that your probe and digest work on a Southern blot of ES cell DNA. The frozen lines in 96 well plates have a limited life span, which does not allow sufficient time for you to work out your detection strategy after the fact.
     If the Southern blots identify targeted lines, those cell lines will be thawed, expanded, their chromosomes counted and DNA will be prepared for further verification of the targeting event. Because locally duplicated sequences can result from targeted insertion, targeting should be verified with an probe outside the targeting vector on the other side.
     Additional information about the detection of targeting events has been excerpted and modified from LePage and Conlon, Methods Mol Med 2006.

Why were there no targeting events?
     It is not clear why some vectors target and others do not. If your vector is within the parameters given above, choosing different pieces to construct a second vector is recommended, although there is little guidance that we can provide beyond recommending that the amount of homologous DNA be increased. Moving the arms to different parts of the gene, and flipping the orientation of the selection cassette sometimes help. It is better to invest your time in building a second vector than to screen more cell lines with a vector that doesn't target at an appreciable frequency.

• An account number.
• A map of the targeting strategy showing the selectable markers, arm lengths, and deletion length. The map should clearly indicate the linearization site.
• Arrange a meeting with us to discuss the targeting strategy.

• We will provide you with ES cell DNA.

• An autoradiogram of a genomic Southern of ES cell genomic DNA cut with your diagnostic enzyme and probed with your external probe.
• For targeting with vectors containing loxP sites, evidence that the loxP sites are functional in the targeting vector.
• 1 milligram of super-coiled targeting vector DNA at 2 mg/ml in TE.

• We will verify the vector, linearize it, and clean it up for transfection.
• We will electroporate R1 (129) or C2 (C57BL/6) ES cells with your targeting construct and select drug-resistant cells. For a generic targeting strategy like that depicted above, we will do six electroporations. From this we expect to pick between three to five 96 well plates of cell lines in duplicate. One will be used to prepare DNA, the other will be frozen.
• Cell line DNA will be provided in 96 well plates to be digested with restriction enzymes directly.
• It is expected that between 200 to 350 cell lines will grow, retain stem cell properties, and give a signal on Southern blots.

• You will cut the DNA and perform Southern blots to identify targeted lines. This must be completed within two weeks of receipt of the DNA.

• We will thaw, expand and refreeze targeted lines, prepare genomic DNA and count chromosomes.

• We monitor the cell culture, electroporation and targeting through analysis of the frequencies of single and double drug resistant colonies, and the morphology of the cells and we target with a control vector in parallel to provide complete quality assurance. However, because of the extreme variability of targeting frequencies (from 1 in 4 to undetectable with similar constructs), we cannot guarantee targeting or provide relief from billing. If all parameters are within the recommended limits and targeting does not occur, the best recourse is to build a new targeting vector and begin again.

• $5,000 per construct in R1 (129) ES cells
• $7,000 per construct in C2 (C57BL/6) ES cells

• We ask that you acknowledge contributions of the Case Transgenic and Targeting Facility in seminars and publications.