southern blot question

[phillipw]

I have done some Southern blot for knock-out mice, including heterozygous and wild type.
My wild-type band (about 13 kb) and mutant band (about 4 kb) co-exist in heterozygous, while wild-type band is only in wildtype.
The question is from one of my collegues, who noticed that the 13-kb band is always much more intensive than the 4-kb band in hets. This ratio seems to be not reasonable since the same probe (shouldbe the same intensity) binding to the same molar amount of target DNA in heterozygous.
Anybody with experience in Southern blot can explain why?
Thanks.

[knockout maker]

Hi there,
This question is a classic one. This band comes from, in the best of the cases, embryonic fibroblasts genomic DNA, alternatively, you targeted an ES cell in S phase (which is most likely), so just one out of four alleles was targeted...in the worst of the cases you have a quimeric clone, contaminated with a negative clone due to random integration or evet WT satellite clones.
Cheers!!

[phillipw]

Thank you very much, Pipette Filler!
I read all your replying messages to my posts.
And I think you are correct regarding several of my questions.
Fortunately recently I solved the problem and the ex-mystery about this "knock-out" mouse turned out to be a transgenic event!
It's a very unpleasant answer, since it means my 6-month work has no way to publish.

By the way, what's "evet WT satellite clones"?

My genomic DNA is from mice, not from ES cells.
So there is no problem of contamination from fibroblast DNA, or something like that.

Again thank you!

[knockout maker]

Hi phillipw!
Oh sorry, I did not notice your genomic DNA came from mouse tails. Well; then I would say that your digest is not complete and knockout alleles remain undigested, mixing up with the wiltype signal. But tell me something, is this the same mice you told it is a transgenic?. If so, you are doing a wrong southern, because I guess your probe is iternal, otherwise you would not detect the transgene. If your probe is still external, then you are detecting a pseudogene. Cheers!!!

Note: WT satellite clones means mixed ES cells clones. After electroporation of your construct in ES cells they are subjected to a week of selection in order to select for recombinants. Sometimes cells that integrated the construct in different sites aggregate and coexist as a single clone during the selection week. After picking one of these and characterize it as a positive, what you will get is a quimeric mice build up on two different ES cell clones, one positive, meaning correctly recombined, and the other negative, meaning random integrant. In the ES southern screening you would expect a more intense wiltype signal. This is quite common indeed. Cheers
Note2: my name is knockout maker, not pippette filler...;o)