Originally Posted by ougadougou
I have sent purified plasmid DNA to mouse transgenic core to develop transgenic mice with that specific DNA piece.
Out of about 45 mice that I genotyped, only about 3 mice gave me the right bands (so I have 3 founders), indicating the presence of the foreign DNA in the mouse. I got few pups from these founders that have the foreign DNA as well. However, few months later, all my genotyping results on new pups from my founders are turning out to be negative (Dont notice any bands). I was confused and re-tailed my founders and genotyped them again. Surprisingly, I dont notice the bands in my founders that I had observed previously. I really doubt that the results I saw on my founders were plain contamination (false positive results). (Note: Not all 45 mice were genotyped at once, may be about 10 mice five different times. Also, when ever I had genotyped, I had a positive control that showed positive results and negative control that showed no bands. So No contamination with my controls). I'm really confused. did any one face the same issues that I did? Also, is there a possibility that the DNA in my 3 founders got integrated in the telomere region, so as the mice got older, the foreign DNA got disintegrated. Any suggestions/answers are welcome. Thanks
It's possible that the DNA didn't integrate until after a few divisions of the fertilized oocyte so that your founders were mosaics (some cells had the transgene, others did not). You may have been unlucky and none of your founders had the transgene in their germline.
It is strange that when you re-genotyped the founders they typed as negative (what tissue did you take?). I guess it's possible that if they are mosaic some parts of the mouse might not contain transgenic DNA. Although you would think that both positive cells and negative cells might both be present in a tissue biopsy. So that's weird.
I would be surprised if all three transgenes integrated at telomeres. Do you have a reason to suspect that the DNA sequence of the transgene might cause preferential integration at telomeres?
Good luck, my experience with transgenic mice made using DNA fragments is that they are a pain in the ass and often give all sorts of random expression patterns. Have you considered making a knock-in or conditionally activated allele at the ROSA26 locus?