southern conflicts with PCR in knock-in mouse screening - why southern positives are not the mutant we want (Jan/09/2007 )
Thanks for clicking this topic!
I am working on a single mutation Knock-in mouse project. Usually southern hybridization is the golden rule to screen the ES cells and first generations of mice. We found some positive Knock-in ES cells. From one of these cells, we even got mice. However, when we amplify the segments flanking the mutation and sequence the PCR product, many of these ES cells and all mice turned out to be wild-type.
Does any one know why this happens? Should I trust southern or PCR in screening?
for me the golden rule is sequencing.
If the sequencing say some is a dud it is a dud. I would trust the sequencing. If you are worried. Re-extract the DNA, do so carefully so as not to contaminate it and run the sequencing again.
And I have seen something a kin to your situation in bacteria and yeast. It is possible that your ES colonies were originally mixed. The mutants were out competent in growth by subsequent expension of the colony during culturing. And when you made your mice, either the mutant ES cells were outcompeted, and only contributed to a small porportion of the mouse, or by this stage your ES cell population was mostly all wild type ES cells.
It is also possible that your gene knock out is somehow lethel to mouse development. Thus no individual can be born with said knock out.
Thanks perneseblue. Your reply is of great help!
BTW, what's a good method to sequence the mutation segment? Should I use PCR for subclone or what?
Yes, I believe you should PCR/sequence the subclones. Making sure everything is pure.
As for seqeuncing the knock in mutation...
Ideally, a complete sequencing over the entire mutation, including some of the flanking wild type DNA would be nice.
Though settling for the junction between the gene and the knock in DNA segment would probably be more practicle.
I am not sure what do you by sequencing method. But since this is genomic DNA, you have to first PCR the region of interest and then sequence the PCR product. (the PCR product may have to be gel purified first, if there are nonspecific amplification)
that's what I asked about
Cause we do the PCR first, and the PCR is done in the mixture of WT/KI, we always got a mixture of bands carrying WT or KI sequence in PCR product.
When I send the purified PCR product to sequencing, the result will be an image with peaks representing incorporation of different nucleotides at the same position.
Usually, at the mutation site, I will get a dominant WT peak, for example, an A for WT, and we tried to mutated it to T. Then in some samples, I can find a small peak of T there. The peak of T, is hardly as high as A in most samples.
Should I expect the T peak to be as high as the wildtype A peak? Should I throw away all ES cells that can NOT yield even peaks of A and T (for ES cells after recombination, the ratio should be 1:1, WT and KI each on a strand of DNA)?
Hope to hear from you again... I am confused by what I get...
Ah, I think this means your colonies are mixed. A hetrozygote ES cell colony should have a near 1:1 ratio. The two signals should be nearly as high as each other in a pure colony
If your mutation is single base pair (umm... I though your knock in was a resistence gene... my mistake).. You should consider using a ARM tetra primer PCR... this system can distinguish single base pair mutations and higher. A hetrozygote will produce 3 bands.. a none specific band, and 2 specific bands, one for wild type and one more for mutant. Comparison between the wild type and mutant band intensity should give some idea of what's happening.
The first thing you should do is colony purify your ES cells.
Using your current data, identify the ES cell population that has the highest mutant concentration of mutant ES cells. Ie the colony which has the strongest signal for the mutant.. the 'T' base pair.
Spead the cells and pick individual colonies. Test them either by ARM PCR, mismatch PCR, or by brute sequencing. Find a colony that has the knock in. One with a 1:1 wild type to mutant signal.
Also, I must ask, do you have conformation that the gene has been correctly targetted? Where the PCR's/southern blots designed corrrectly? Ie you knock in consturst has not actually targetted some other region in the genome, and the southern blot was pulling up this mistargetting rather then the correct targetting. This is always a fear in targetting.