Transgenic mouse - (Aug/23/2007 )
I am making a transgenic mice for the first time.
I need help to get ready with things I will be needing for screening of the pups and afterwards.
I have my construct ready and i know i need to remove the rest of the vector sequence before microinjection. The core facilty will do the microinjection and give us the promising pups for screening.
Can anyone pls let me know the steps after i have the pups in hand and
what all i should beready with?
how to manage the pups...litters...how to breed them , when to start screening ..ect
Thanks for ur hepl.
Aftre the microinjections, you will get a whole bunch og mouse tails from the facility that you will need to screen for Tg. So, before that time, you should have your PCR working for "typing" the tails. You can optimize this PCR on the contruct you have and try to optimize it for very little amount of DNA too.. just do a titration 'cos you want to be able to detect low copy number Tg too. After you have identified some that are positive, you tell the facility to transfer those specific mice to you. Which you start breeding once they are 5-6 wks of age (even in Quarantine if possible) to the strain you want to breed to. For setting up matings, you have to put 1 male mouse with 1 female mouse in a cage (Note about harrem matings later). For matings, mice should be atleast 5 wks of age. Once the female is pregnant, it will drop litter in 3 wks. YOu let the pups stay with mom (&dad if you like) until pups are 3 wks of age. After that, you have to separate them into their own cages, a process called weaning. Depending on what yoru lab follows, you can either type the pups when they are 8-11 days (I believe) or after weaning. For weaning, you put all male pups in 1 cage and female pups in another cage, upto a maximum of 5 in each cage. NOTE: more than 5 mice/cage is overcrowding and not allowed. We usually "ear tag" them at weaning time and take a sample of their tail whihc we digest O/N with proteinase K and use it for PCR next day (or whenever you get to it). So, when you are optimizing your PCR, make sure you use a tail DNA sample from a non-Tg mouse to confirm that PCR is working. Once you have positive pups, you can set those up in next round of matings with fresh partners, which you can probably order from some company liek Jaxson/Charles river etc. After you set up mating, it takes 3 wks for litter to drop, 3 more wks before you wean and 3 more wks before you set up next round of mating/can do expts with them unles syou need newborns for expts. This way you keep backcrossing to the strain you need to get Tg on. ONE MOST IMPORTANT THING: Keep very good records, of mating set up date, date of borth of pups, weaning date, who goes into next round of mating etc 'cos I have had to go back even 4 years to figure out some contamination I got in my mice thanks to acquiring a strain. Also, keep making a note of what backcross generation are you on. Mice are considered fully backcrossed after 10 backcrosses. Until then, you should only cross them to "pure" mice (say B6 or BALB/c) but not with Tg negatives. About Harrem matings: If you need to maximize mice, you can set up 1 male with multiple females (upto 4, 'cos >5 mice/cage is overcrowding) but you have to separate the females out into separate cages before they drop litter again due to overcrowding issues. Also, dpeending on the strain, mice can keep having pups for several generations. This means that if you set up a mating, and got one litter, you wean this litter and around weaning time, the female may drop the next litter. So, you decide how long you want a mating to be around and when you want to refresh matings. Sometimes, 1st litter is terrible, but as mice get older, litter size decreases too. If your Tg is expressed and you can confirm expression by FACS, go for it but don't euthanize any mice you get from microinjection facilit for this or any other reason until you get some pups that are carrying the Tg and you are sure they are expressing. Another note: You may get say 4 different pups from microinjection facility. Keep them all and as separate lines of the Tg. Cross them all to next generation and look for "transmission" of Tg into next generation. Some lines may not transmit or express. Oh, when to start screening for expression... if you have to do this in blood, you can start at ~5 wks of age.. even sooner if desperate. I never have time to do this sooner. This is all I can think of right now. Good luck.
1. test the tail tips for the right tg mice. (PCR for a sequence only in the transgene).
2. start breeding.
3. keep records.