11 replies to this topic

[linboren]

can any body help me with this:
The project i'm working on is to breed some transgenic -/- mice for experiments.
i started from putting my +/- male and female mice together to generate -/- animals, but so far i got only +/+ pups. Theoratically if it is embryonic lethal, i should have 33% +/+ and 66% of +/- instead. is there any other reason that i can't get -/- offspring?
thank you.

[Rnotk]

This might not be an answer that you are looking for, but

I had exactly same situation few months ago, and I could not figure our for a while.
and it turn out to be the genotype of parents were wrong (mother was wt, instead of het).

I am not blaming undergrad who did the genotype for me, but I realize it is important for me to make sure everything by myself.

[bob1]

Another option is that the -/- is embryonic lethal or biased in some fashion such that mendelian ratios don't work.

However I agree with Rnotk that it is quite likely that the genotype is wrong, I have seen this happen several times in a few different labs.

[linboren]

bob1, on Jun 16 2009, 08:31 PM, said:

Another option is that the -/- is embryonic lethal or biased in some fashion such that mendelian ratios don't work.

However I agree with Rnotk that it is quite likely that the genotype is wrong, I have seen this happen several times in a few different labs.

say if the genotyping thing was correct... is it possible the eggs and sperms for some reason were not able to carry mutant allele so that only +/+ zygotes were produced?

[bob1]

It is possible that mutant eggs or sperm are not viable, but then you wouldn't get +/- genotype either, unless it is specific to only one type of gamete (e.g. is only lethal to sperm).  It is much more likely that the foetus is not viable as a -/-.

[Carlton H]

I think we're missing the point here...

The question is why does he NOT see heterozygotes in his litters.  It's not just that the -/- are not viable, but the +/- are not either.

I think the likely answer is: Your mutation is dominant-negative.  The product of your mutated gene is interfering with the normal function of the wild-type gene, rendering all your hets inviable.

Cheers,
-Carlton

[Chakchel]

Carlton H, on Jun 19 2009, 05:18 PM, said:

I think we're missing the point here...

The question is why does he NOT see heterozygotes in his litters.  It's not just that the -/- are not viable, but the +/- are not either.

I think the likely answer is: Your mutation is dominant-negative.  The product of your mutated gene is interfering with the normal function of the wild-type gene, rendering all your hets inviable.

Cheers,
-Carlton

...but then, where did he get his +/- male he started with?

I guess another check of the genotype would be the best to start with.

[linboren]

is it possible the mutant allele can be wiped out in the C57BL/6J background, but not in the chimeric mice since they are more "artificial"?
in this case we might have hetero only in the 1st generation.

[Carlton H]

Chakchel, on Jun 22 2009, 02:41 AM, said:

...but then, where did he get his +/- male he started with?

Touche.  Haha...

@linboren - are you moving the mice onto a different background at all?  I was previously working with a mutation that was lethal in blk/6, but not in balb/c.  Could this be a similar issue?

[linboren]

Carlton H, on Jun 25 2009, 11:07 PM, said:

Chakchel, on Jun 22 2009, 02:41 AM, said:

...but then, where did he get his +/- male he started with?

Touche.  Haha...

@linboren - are you moving the mice onto a different background at all?  I was previously working with a mutation that was lethal in blk/6, but not in balb/c.  Could this be a similar issue?

the mutation was generated in chimeric male, then transferred to the C57BL/6J background.

[Animaniac]

Are they Tg or Knockout mice? (here in Aust we tend to use -/- for KO as it represents null)? However if you are not transferring onto a different background, and you say you have het parents and these mice have bred previously and all you get is +/+, then according to simple mendelian genetics, your parents would have to be both +/+.

I would check your primers and re-test your parents.

Good luck

[blackmouse]

My question is on which chromosome is the knock out mutation???
If it's on the X chromosome your males are hemizygote and potentially sterile...happened to me.
Either due to the mutation or just simply since you disrupted the X chromosome where a lot of spermatozyte maturation genes are encoded.
My bet..if it is on the X ..check by IHC for mature spermatocytes!!
Good luck!