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)
>
>
>