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.

Genetic Background and Gene Targeting
     In the past, most ES cell lines used in gene targeting had the 129 genetic background. Formerly, if a mutation on the C57BL/6 background was desired, the mutant mice were crossed nine times or more to C57BL/6J mice to establish it on that strain. Recently, a C57BL/6 cell line which targets and transmits well was established, the C2 line. This line has a C57BL/6NTac background, which although not identical to the commonly used C57BL/6J, is closely related, diverging as a separately maintained colony in 1951. Mutants targeted in C2 can either be maintained on C57BL/6NTac by crossing chimeras to mice purchased from Taconic, or can be crossed to C57BL/6J mice purchased from The Jackson Laboratory. The C57BL/6NTac genetic background of C2 is more closely related to C57BL/6J than that of the Bruce4 cell line (Hughes et al 2007 (.pdf)) which is commonly used as a C57BL/6 ES cell line. If your experimental goals require studies of mice on a C57BL/6 background, we recommend that you choose to target in C2 cells.

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 same strain of mice as the ES cell line. Small differences in DNA sequence can have very large effects on targeting frequency. 129 genomic clones in BAC vectors (.pdf) for targeting the R1 cell line 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. C57BL6J genomic DNA BAC clones for targeting the C2 cell line can be identified with the UC Santa Cruz genome browser and purchased from BACPAC Resources Center CHORI.
     Alternatively, DNA from the "wrong" strain can be used to construct targeting vectors if there are no differences in sequence--single nucleotide polymorphisms (SNPs) are distributed in a mosaic pattern in mouse strains, with blocks of high sequence difference separated by blocks of identical or nearly identical sequence. The locations of sequence differences for your region of interest can be ascertained here on the Jax web site.
     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.
     We also offer targeting vector design and construction services.

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.

• From the start date of gene targeting, it will be a minimum of 5 weeks before the first set of cell line DNAs is provided, and a minimum of 3 weeks after positive targeting events have been identified by Southern blotting before we will deliver the second set of DNAs for confirmation of the selected, targeted lines.

• 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 vectors with loxP sites, sequencing of the loxP sites in the targeting vector to show that they will be functional.
• 500 micrograms 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 two electroporations. From this we will pick two 96 well plates of cell lines in duplicate, one for DNA and one for freezing.
• Cell line DNA will be provided in 96 well plates to be digested with restriction enzymes directly.
• It is expected that about 170 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 promptly (~2 weeks) after receipt of the DNA to ensure recovery of cell lines.

• 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. 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.

• $5,500 per construct in R1 (129) ES cells (Nagy 1993)
• $7,700 per construct in C2 (C57BL/6) ES cells
• $7,700 per construct in PC3 (129, protamine-Cre) ES cells (O'Gorman 1997)

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

• Although the IACUC and IBC/rDNA do not regulate gene targeting in ES cells, the generation of chimeric mice with any genetically manipulated ES cell line is regulated by both bodies. Approval of protocols by these committees typically occurs at monthly meetings after submission, review and revision of protocols. Therefore, work to obtain approval from these committees well in advance. The regulatory requirements for generating chimeras are here.

You will need the following information to place an order

• Account Number
• A .pdf file of the targeting construct