1. Published studies by one group suggest that the minimum size for a targeting arm is approximately 0.5 kb, however, this size targeting arm will produce very low efficiency homologous recombination. The contributors of this protocol generally use a minimal targeting arm length on one side of 1 kb, with at least 2-3 kb on the other side. There is no reason why the two targeting arms can't be of approximately the same length. They generally make both the targeting arms approximately 2 kb in size (see Hint #1).
2. The contributors of this protocol frequently PCR the targeting arms using genomic DNA derived from RW-4 (129/SvJ) ES cells, or from cloned genomic DNA, if it is from a 129/SvJ source (see Hint #2 and #3). A few PCR errors seem to cause few (if any) problems for efficient homologous recombination. PCR certainly makes the ends of fragments easier to manipulate for insertion into vectors containing PGK-Neo.
2) Single vs. Double Selection Techniques
1. Most targeting constructs in the past have used both PGK-Neo and HSV-TK to perform a positive/negative selection. However, gancyclovir treatment of ES cells is quite toxic and the contributors of this protocol find that it negatively affects the ability of ES cells to go germline. They are willing to accept the lower rate of homologous recombination in exchange for a better rate of germline transmission. Therefore, they use single-selection vectors with PGK-Neo exclusively. Several different kinds of PGK-Neo cassettes are available.
2. The contributors of this protocol are now routinely using PGK-Neo that is flanked by Lox-P sites, so that the PGK-Neo selectable marker cassette can be removed by CRE-recombinase to avoid potential problems with "neighborhood effects" of the PGK-Neo cassette. Recent work from their laboratory and others have suggested that this neighborhood effect is very problematic in multigene clusters (see Citations #1 and #2). If the gene of interest is in a multigene cluster, you will need to anticipate the potential neighborhood effects of retained PGK-Neo cassettes. Lox-P flanked PGK-Neo cassettes and a high performance CRE-recombinase expression plasmid (pTURBO-CRE) are available from the contributor of this protocol (see Protocol for Removal of Selectable Marker Cassettes from Transfected ES clones using Cre-Lox Mediated Recombination).
3) Detection of Homologous Recombination Events
1. Although some people have successfully used PCR to detect homologous recombinants, the contributors of this protocol recommend against this practice. They strongly recommend that you develop a unique probe that is external to the targeting sequences themselves and use it to screen using Southern analysis. It does not matter whether this probe is upstream or downstream from the targeting construct, it only matters that it is completely external, and that it contains no repetitive DNA elements.
2. In addition to defining a homologous recombination band, Southern analysis also allows for the assessment of the molarity of mutant bands, which is difficult to do by PCR. If the wild-type and mutant bands are not equal in intensity, you must be suspicious that the targeted clone is contaminated with wild-type ES cells. If it is, the wild-type cells will have an advantage when they are injected into blastocysts, and the result will be chimeric males that throw mostly wild-type agouti pups. This can be a huge problem. If the targeted bands look correct on the Southern, but the molarity is suspect, then the contributors of this protocol strongly recommend that the cells be re-selected in G-418, or even replated by limiting dilution to subclone the ES cells of interest.
4) Generation of Chimeric Mice
1. There are several services available for making chimeric mice from the targeted ES clones (see Hint #4). Commercial and Academic Injection Services can be accessed from the "Links" section of the contributor's website.
Protocol Tips
1. There may be a significant advantage in using substantially longer targeting arms; however, large constructs are more difficult to manipulate. In the contributors' experience, targeting arms of approximately 2 kb on each side will yield homologous recombination efficiencies of 1 to 2%, which are adequate for most purposes.
2. The contributor's embryonic stem cell line was isolated from 129/SvJ blastocysts and is called RW-4. A recent paper by Elizabeth Simpson and colleagues at the Jackson Laboratory (see Citation #3) has carefully evaluated genetic variation among all of the 129 substrains. The contributors of this protocol strongly recommend examining this paper before starting any experiments. The wide variety of embryonic stem cell lines that can be obtained from different investigators can be highly divergent within the 129 background. Choose these cell lines with care, since they are not all equivalent. There are a number of allelic differences between the 129 substrains that cause minor histocompatability differences, and there are many differences in the backgrounds of the 129 substrains (and even in ES cell lines obtained from these strains).
3. Of note, a careful evaluation of the 129/SvJ substrain (Jackson Laboratory #000691), from which RW-4 cells were made, reveals no allelic differences between the mice and the RW-4 cells. Therefore, no obvious mutations have been acquired during the preparation of the contributors' cell line.
4. Please note that you must have an active animal protocol that covers the chimeric mice that you will be receiving on file before injections can begin.
