puzzling knockout observation.

[phillipw]

We ve got a puzzling knockout observation.
Right now I would not like to call it the phenotype since we haven't figured out what's going on.

So let me tell you the short story.

A previous postdoc in the lab made a knockout mouse by using a very simple construct (2.1kb)5arm-pGKpuro-3arm (3.7kb). He screened the transfected ES cells by using PCR. Then chimeric mice were obtained. After breeding, heterozygous mice were obtained. Next should be to get the homozygous.

But a wierd thing is that not only homozygous mice were not be able to obtained (supposedly the knockout is lethal) but also the wild type has much fewer than expected. The hetero : wildtype ratio is 9:1.
It is normal that knockout is lethal for homo . But it s hard to believe that the wildtyp is also lethal after hets cross with hets.

By the way, when we cross hets with WT, the ratio is normal, i.e. 50:50.

Anybody out there knows what s going on for this knockout mice?
Or anybody has ever encountered this kind of problem.

[knockout maker]

Check whether your gene is imprinted. You might be selecting against wildtypes if your knockout allele is the one imprinted, which in turn depends on the parental origin. It might happen with some heterozygous matings but not with others depending from which of the parents the knockout allele has been inherited.
Cheers,

[phillipw]

Thanks for reply!
But I think if there is an imprint, the bias should be against heterozygous since the only wild type allele may be printed in hets and render it to be like homozygous.
For wild type mice, imprinting will render them to be like heterozygous.
The reason should be something else.
Unfortunately, I m still looking for the reason.

[eudora]

you may have considered this:
Is it possible that the mother may have cannibalized the new borns (before you had a chance to see them), and it just happens that they must have all been Wild types ?

[knockout maker]

Hi there!
Well let´s think in green. How sure are you that the knockout was successful?. Could not it be that your posdoc screened with internal primers and you are segregating just a transgene randomly integrated?. Sometimes people do not fully understand what means "external primer"...Cheers!!

[phillipw]

You got it!
The previous postdoc even didn't screen the ES by using Southern, but by PCR.
That 's pitfall and we did pay the price for that.
Because of that, we didn't get the real knockout, instead, we got a transgenic mouse.
My Southern and genetic data (from breeding) showed it is a transgenic mouse.