Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Knockout animal mating


  • Please log in to reply
5 replies to this topic

#1 SMN

SMN

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 16 April 2009 - 07:39 AM

In order to save the time for genotyping, I would like to mate my knockout animal so that all their offspring will be knockout too. But I know other lab only mate between heterozygote as they said the knockout effect can be more sustain without logical explaination. Can anyone explain? Which method do you routinely used? Thank you!

#2 Carlton H

Carlton H

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 95 posts
0
Neutral

Posted 17 April 2009 - 11:19 AM

I'm not sure I totally understand your question, but I get the idea and have some advice...

Yes, if you have viable double-knockouts you can inbreed them to avoid phenotyping, however I do not recommend this, for the same reason that inbreeding is never recommended in general: you're going to bring out all the recessive, deleterious phenotypes and may in time have problems with inbreeding depression, a highly inbred state which is characterized by poor health and poor mating efficiency. Plus, inbreeding is generally considered to be poor practice and your advisor / boss / supervisor will just think you're lazy, and that's never a good thing, either.

In other words, inbreeding will save you time now, but it'll hurt you in the long run. Don't take the shortcut, and just outcross your KOs.

Cheers,
-Carlton
NextAdvance
Reliable laboratory instruments for the life sciences, designed to enable you to work more effectively and increase productivity. Check us out!

If my answers help you, please take a moment to check out our site and see if any of our products would help you as well! Thanks!

#3 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 03 May 2009 - 02:10 AM

Many times, the KO animals are sterile or donot mate properly or donot have a good sized litteror the fetuses get aborted. So in these cases one would have to mate heterozygotes to have KO animals.

#4 Animaniac

Animaniac

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 07 July 2009 - 07:58 PM

In the interest of the 3R's, you should if possible create a homozygous animal. Inbreeding depression happens after more that 20 generations. If you need hets for control then only breed them when you need them.

If these mice are on a specific background, then you only need to cross once every 4 generations to maintain the background.

If there are other problems with a homozygous animal such as what scolix has described, then you may need to keep as het, otherwise it is not a case of laziness, but of reducing un-usable animals un-necessarily by breeding het to het.

Remember..........reduce, reduce, reduce

#5 Mighty Mouse

Mighty Mouse

    Squeak!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 124 posts
3
Neutral

Posted 12 September 2009 - 09:39 AM

In order to save the time for genotyping, I would like to mate my knockout animal so that all their offspring will be knockout too. But I know other lab only mate between heterozygote as they said the knockout effect can be more sustain without logical explaination. Can anyone explain? Which method do you routinely used? Thank you!



We have a number of KO animal lines in our lab and we always mate between Hets. There are a number of reasons why this is better than mating KOs and WTs separately:

1) The primary reason is that if you want to compare KOs and WTs to one another experimentally you want to ensure that the differences between them are limited to their differences in genotype and not other factors such as how the dams interact with their pups or potential in utero differences bewteen KO and WT dams. We do a lot of behavioral work in our lab and dam-pup interactions are known to have a variety of effects on the central nervous system. I imagine there are likely effects on other systems as well. Thus, it is ideal to be comparing litter-mates to one another that are raised as similarly as possible to ensure any differences observed are most likely due to the differences in genotype.

2) I imagine there is some potential for a little bit of genetic drift and that your KO and WT populations may begin to become separate populations of mice that may have subtle genetic differences, particularly if they have not yet been backcrossed and you're using F1 hybrids. For example, I was asked to do some tests on some KO and WT animals from a different lab. There was an effect of the KO on our task, but all the KO animals had agouti coat colors and all the WTs had black coat colors (they were originally derived from a C57BL/6 x 129/SV crossing). Thus, my conclusion could have just as reasonably been that the coat color of the mice was the cause of the difference as opposed to the KO ;) ! if the coat colors are different then what other genes are different? Probably a lot! Now I have to take the time to backcross these mice on to B6 for 5 or so generations because this other lab is too lazy to breed their mice properly :( . Moral of the story: don't take the short-cut! It'll hurt the interpretation of your experimental results in the long run.
We are all artists...painting with experience on the canvas of life

#6 Animaniac

Animaniac

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 13 September 2009 - 07:18 PM

I was just reading Carlton's reply about the dangers of inbreeding................ I must have been very distracted when I looked at this topic the last time as I didn't address this comment.

DO NOT outcross your KO mice!!!!!!!!!!! I cannot stress this enough. It is not common practice among ANY animal facilities world wide. Although some researchers may need to use outbred mice, the absolute majority of people in the world use inbred mice.

This is not BAD PRACTICE, but absolutely necessary to reduce variability among results.

If you look at any reputable facility around the world that supply mice for research you will see for yourself.

All strains such as Balb/c, FVB/n, C57BL6/J are all inbred mice.

Knockouts are generally back-crossed for 10 generations onto a strain such as C57 or Balb/c to maintain their inbred status.

As for Mighty Mouse's comments, is true, you may need to breed hets if you need hets for controls, we achieved this by crossing a female knockout with a WT male. All pups are then hets, no genotyping necessary. This way you still maintain the colony as homozygous and only breed hets when needed. Thus reducing numbers. This also addresses the issue regarding the dam's behaviour toward the young as she will be a knockout too. Just another option to consider. Also reduces the invasiveness toward the animals as no genotyping is needed, this is the main reason our researchers chose this method.

Genetic drift occurs after 4 generations of random breeding. Also the laws of nomenclature require that after a certain amount of inbreeding within a facility, then the mouse must change it's name tag at the end to the facility it is at. Example: If you breed C57BL6/J mice, the J stands for Jacksons Laboratories where these were first bred. If you are breeding your C57BL6/J mice for a certain number of generations (sorry can't remember just now) then you should publish your animals as C57BL6J/yourfacilitycode. This is to alert others who read publications that your mice were genetically different to the true Jacksons mouse. This is explained on the Jacksons Laboratories Web site.

But once again I cannot stress enough that inbreeding in mice is the norm and very few research is done on outbred mice. Your supervisors would be very angry if you just went ahead and started outbreeding their knockout mice without checking with them first.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.