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 are 5 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.
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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 can be truly sure of
your positives.
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.
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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.
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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 control plasmid
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
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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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