Transgenic--how to establish the mouse colony - (May/27/2008 )
Thanks to all of you for the information, "cellcounter" the links were very useful..
I have got a beautiful gel after PCR, now i am ready to make crosses between mouse which showed a right size band, my question is how to make crosses between positive founder pups for establishing the transgenic mouse colony?
I have positive 1-male and 4 females out of 23 pups. I had marked the ears so I can put
a) cage1- positive male with 2 non-transgenic females, once females are pregnant, i can seperate those females
cage 2-5: positive 4 females with one non-transgenic male ecah in 4 cages
1) Is my thinking correct?
2) can I use the non-transgenic pups seen in my PCR genotyping for mating ?
3) how to check that the mating is done and a female is pregnant?
4) Can I mate positive male with a positive female at this point ?
Thanks for the help !
1. It is good to segregate transgenic alleles to the point that each subsequent generation gets the same gene dosage. So, I generally mate the founders with wt Bl6, and (sometimes other inbred backgrounds too), and after a few generations, depending upon your patience, you can start breeding hemizygous.
2. I would not use non-transgenic pups. Just use pure inbred mice from Jackson or Charles river.
3. Female gets plugged, which you can check the next morning, but in this case, just leave them alone for a few weeks!
2. You know what, this entire matter requires a lot of considerations, that I can not write them all here. So, I would suggest that you read some about breeding up a transgenic colony and then ask some practical questions that are not covered.
http://search.vadlo.com/b/q?sn=158621799&a...ding+&rel=0
3. You may also want to check out some OXford/Humana press books that I am sure should be available in your library. There are just too many things involved here for me to write-up.
2. I would not use non-transgenic pups. Just use pure inbred mice from Jackson or Charles river.
3. Female gets plugged, which you can check the next morning, but in this case, just leave them alone for a few weeks!
2. You know what, this entire matter requires a lot of considerations, that I can not write them all here. So, I would suggest that you read some about breeding up a transgenic colony and then ask some practical questions that are not covered.
http://search.vadlo.com/b/q?sn=158621799&a...ding+&rel=0
3. You may also want to check out some OXford/Humana press books that I am sure should be available in your library. There are just too many things involved here for me to write-up.
Thanks again, I am reading from this site itself, I will check for this book surely.
Usually its suggested to check for the plugs the next day after setting up the mating, is there any particulat reason for leaving for few weeks in my case?
How long does this plug remains visible?
Is Bl6 , B6 same as C57BL/6 strain?
Thanks so much !
Usually its suggested to check for the plugs the next day after setting up the mating, is there any particulat reason for leaving for few weeks in my case?
You check plugs when it is important for you to dissect out embryos at a precise stage in gestation. Say e9.5. In your case, you are only interested in the next generation, so it doesn't matter whether you know it the next day or in three weeks. Anyways, seeing plug does not always gurantee there will be a viable pregnancy and pups.
How long does this plug remains visible?
Generally you see them first thing the next morning of your setting up matings. If you wait too long, say afternoon of the next day, some plugs may fall off by that time, and you have no way to tell.
Is Bl6 , B6 same as C57BL/6 strain?
Yes.
Thanks so much !