GENOTYPING REQUIREMENTS FOR GENE TARGETING (I. page 1-3) AND TRANSGENIC EXPERIMENTS (II. Page 4-5)

I. For Gene Targeting:

A successful gene targeting experiment is absolutely dependent on perfect genotyping assays worked out before transfections are begun. Please read the following discussion carefully and then make an appointment with Tam to discuss your genotyping strategy.

NOTE: It takes as much or more effort to get the genotyping assays actively working as it does to make the targeting vector.

The following discussion focuses on targeting experiments that include “remote” mutations as defined below, but is relevant to all targeting experiments.

“Remote” mutations are desired targeted mutations that are in targeting vectors in addition to the neomycin (or other positive drug) selection cassette and that have genomic sequences between the positive drug selection gene and the mutation.

Genomic rearrangements

There are5 possible genomic integration events that can occur upon transfection/selection and that we will concern ourselves with. You want only the one (category #3 below/and sometimes #5 for controls) and must design sufficient genotyping assays to determine exactly the cells in which the exact correct/desired event has occurred.

  1. Random integration anywhere in the genome/not at your desired locus - don’t want these and are usually easy to rule out by even just one genotyping assay.
  2. Insertion of your targeting allele into the desired locus resulting in an imperfect duplication. You get one copy of the wildtype with an adjacent copy of your mutant allele. This can happen on either side of the wildtype locus (5’ or 3’) - don’t want these, and you can be easily deceived by these if you have incomplete genotyping info.
  3. Correct homologous recombination of your targeting allele resulting in a replacement of the wildtype locus with your targeting allele including your “remote” mutation - these are the ones you want, but they can easily be confused with category 4 (see below).
  4. Correct homologous recombination of your targeting allele resulting in a replacement of the wildtype locus with your targeting allele NOT including your “remote” mutation but also WITH A RANDOM INTEGRATION somewhere else in the genome of at least the part of your targeting vector with your remote mutation - these clones are probably rare, but you really don’t want the uncertainty that what you think is correct is really one of these. You can easily be deceived into thinking these are category 3 (see above) if you have incomplete and/or improper genotyping assay.
  5. Same as 4 but with no additional random integration event. These are good wildtype control lines in that they will have the neo gene but not your “remote” mutation. These can be confused with category 3 clones if you do no genotyping for the remote mutation.

When all these events are individually screened by a collection of complimentary genotyping assays then you canbe truly sure of yourpositives.

Genotyping requirements

  1. Southern and/or PCR assays to assess proper recombination of both the 5’ and 3’ ends of the targeting vector. You absolutely must have Southern probes/PCR primers that are homologous to DNA sequences not contained in the homologus arms of your targeting vector. These “external” (as opposed to “internal” or within the targeting vector) probes/primers are absolutely necessary for demonstrating unique homologous recombination into the proper locus. You need assays that demonstrate homologous recombination of both the 5’ and 3’ ends of the targeting vector. This usually requires sets of probes/primers external to both the 5’ and 3’ ends, but on rare occasions can be done with one probe if the detected band includes the entire targeting vector. Having assays for both the 5’ and 3’ ends is the only way to rule out the insertion/duplication event described in #2 above.
  2. For “remote” mutations, a definitive assay to demonstrate homologous recombination including the remote mutation to rule out #4 and #5 above. Do not depend on the remote mutation “tagging” along with the drug selection event.

Before we embark on a project, we require that people demonstrate on DNA from wildtype ES cells (we can supply this) that all the planned genotyping assays in fact work. You will need to show us that:

1.Restriction enzymes digest wildtype ES cell DNA.

2. Southern probes give a single wildtype band on digested ES cell DNA.

3. PCR reactions work at the single copy level in the presence of complex genomic DNA. These reactions usually involve amplifying several kilobases of DNA. We have seen these work for greater than 5 Kb. See Tam for contacts about working out conditions. (You may need to make positive control plasmids for testing PCR reactions - CAUTION – make the control amplified band a detectably different size than your real recombinant band - because when you screen your ES clones, you don’t want to be uncertain about whether you are looking at a real recombinant or contamination with your control plasmid. An alternative approach to making a positive control plasmid involves designing primers that span the neomycin gene- ie. one external primer and one internal primer in the opposite homologous arm sequence. Demonstration that these primers work on wildtype DNA does not guarantee that they will work on the longer homologous recombinant DNA, however). Please note that lox sites will not serve as amplification primer sequences (too AT rich) so it is usually imperative that a novel restriction enzyme site is engineered at a remote lox site or any other remote mutation for that matter.
Beware of repetitive DNA in your targeting arms and probes.

II. For Transgenic Experiments

A successful transgenic experiment is absolutely dependent on perfect genotyping assays worked out before injections are begun. Please read the following discussion carefully and then make an appointment with Tam to discuss your genotyping strategy.

NOTE: It can take as much effort to get the genotyping assays actively working as it does to make the transgenic construct.

Genotyping requirements

Southern and/or PCR assays to assess integration of the injected transgenic construct into genomic DNA.

Before we embark on a project, we require that people demonstrate on wildtype tail DNA spiked with positive control template that at least one planned genotyping assay works. This can be either a Southern or more likely a PCR assay. For both, the positive controlplasmid DNA must be titrated down to the number of molecules that are equivalent to the number of molecules of a single copy gene in the amount of genomic DNA used in the assay.

For Southerns:

You will need to show that:

1. Any planned Restriction enzymes digest wildtype tail DNA.

2. Southern probes give a single wildtype band on digested tail DNA spiked with positive control template.

For PCR assays:

You will need to show that:

PCR reactions work at the single copy level in the presence of complex genomic DNA. CAUTION – it is often advisable to make a positive control template that will give an amplified band that is a detectably different size than your real integrated band - because when you screen your pup tail DNA, you don’t want to be uncertain about whether you are looking at a real integrant or contamination with your control plasmid. Please note that lox sites will not serve as amplification primer sequences (too AT rich).

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