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can't get homozygous mice, why?


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11 replies to this topic

#1 linboren

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Posted 12 June 2009 - 09:58 AM

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.

#2 Rnotk

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Posted 15 June 2009 - 10:33 PM

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.

#3 bob1

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Posted 16 June 2009 - 04:31 PM

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.

#4 linboren

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Posted 17 June 2009 - 07:08 AM

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?

#5 bob1

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Posted 17 June 2009 - 04:29 PM

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 -/-.

#6 Carlton H

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Posted 19 June 2009 - 07:18 AM

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
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#7 Chakchel

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Posted 21 June 2009 - 10:41 PM

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.

#8 linboren

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Posted 22 June 2009 - 01:55 PM

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.

#9 Carlton H

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Posted 25 June 2009 - 07:07 PM

...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?
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#10 linboren

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Posted 30 June 2009 - 04:07 PM

...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.

#11 Animaniac

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Posted 07 July 2009 - 07:35 PM

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

#12 blackmouse

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Posted 08 June 2010 - 10:54 PM

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!




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