David

Of the mutants I have made, I always get a fair percentage of putative
KO's that are a result of a single crossover event when I use non-purified
linearized targeting vector. Far fewer is the targeting portion is
excised.

Single and doubles are identified by multiple PCR reactions with a series
of primer pairs designed to amplify product from a single-copy gene
scenario in less than 1pg of gDNA.

This is probably obvious, but the general screening primers I use to
identify putative KO's are:

P1- amplifies the 5' targeting arm (result is the same band size seen in a
control PCR of the targeting vector used) - also identifies potential
rearrangement during the recombination event as you get smaller or larger
bands.

P2- is the 3' version of P1

P3- amplifies the entire Bsr cassette using a forward primer that touches
down on the extreme 3' end of the 5' targeting sequence and a reverse that
primes the extreme 5' end of the 3' targeting sequence - this reaction
amplifies the Bsr cassette in a control PCR using the targeting vector and
a band of the same size in the putative KO's. It will amplify a smaller
region (on average 500bp) in WT cells. This is the sequence that will be
knocked out if a double recombination event occurs.

P4- amplifies a product derived from sequence upstream of the 5'
homologous targeting sequence and a reverse primer in the Bsr cassette. No
product in WT cells. No product (or possible larger product) if the KO
contains only a 3' recombination event. Also serves to assess integrity or
re-arrangement of sequence upstream of targeting sequence.

P5- is the 3' version of P4. Identifies the 3' recombination event. No
product in WT or if a single 5' recombination event occurred.

P6- Forward primer touches down in the sequence that should be KO'd from a
double recombination event and the reverse touches down in the 3'
targeting sequence. A product is obtained in WT cells and single
recombination events.

P7- reverse version of p6 primers.

P8- control primers. Amplify a non-related gDNA sequence that is a length
similar to your largest expected amplicon in the KO screen. Just serves as
a positive PCR control to ensure your gDNA is PCR friendly. All samples
should contain this amplicon and are suspect if not there.

Whenever all screening reactions have been performed, it's almost never
ambiguous to identify your double crossover KO's which can be then further
verified by Southern if necessary, RT-PCR and western if Ab's are
available.

Joanne Kilford wrote:
> David
>
> I have just had exactly the same problem trying to generate a knock
> out in the same way. I cloned 5' and 3' non-coding DNA either side of
> the Bsr in the LoxP vector. Initial PCR results indicated a knock out
> but upon further investigation I also came to the conclusion that I
> had a single crossover recombination event.
> I have now designed a new construct in which I have only deleted
> certain domains within the protein that we know are required for its
> function. I have therefore been able to design 5' and 3' arms out of
> the coding DNA. I have cut out the insert, purified it and transformed
> the cells and (I don't want to speak too soon!) but appears that I may
> have a double cross-over recombination.
> I don't know if you are able to take a similar approach but I hope it
> can be of some help!
>
> Jo
>
>
> Quoting "David Knecht" <[log in to unmask]>:
>
>> We have been trying to knock out a gene and have a strange result.
>> The construct is standard with 5' and 3' non-coding flanking region
>> DNA cloned upstream and downstream of Bsr in the LoxP vector.  We cut
>> out the insert, purified and transformed cells.  We got what appeared
>> to be knockouts based upon PCR screen with internal and external
>> primers (we don't have antibody yet).  However further analysis gave
>> us some anomalous PCR bands.  The best explanation for what we have is
>> a single crossover recombination of a circular version of our linear
>> fragment into the 5' homology region of the gene.  Has anyone seen
>> this before?  Any suggestions as to how to prevent it so we can get
>> the double cross-over gene deletion?  Thanks- Dave
>>
>> Dr. David Knecht
>> Department of Molecular and Cell Biology
>> Co-head Flow Cytometry and Confocal Microscopy Facility
>> U-3125
>> 91 N. Eagleville Rd.
>> University of Connecticut
>> Storrs, CT 06269
>> 860-486-2200
>> 860-486-4331 (fax)
>>
>>
>>
>


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Michael A. Myre, PhD
Center for Human Genetic Research
Massachusetts General Hospital
Harvard Medical School
Richard B. Simches Research Center
185 Cambridge St., CPZN 5.821A
Boston, MA  02114
617-643-5536
[log in to unmask]
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