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Problem with Transgenic Mice

Problem with Transgenic Mice - Knockout Mouse Forum

Problem with Transgenic Mice - Discuss and post questions about knockout mice, transgenic mice and other transgenic animal models.


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  #1  
Old 09-30-2011, 04:45 AM
Pipette Filler
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Default Problem with Transgenic Mice



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
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Old 10-17-2011, 06:35 PM
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Default Re: Problem with Transgenic Mice

Quote:
Originally Posted by ougadougou View Post
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?
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Old 10-18-2011, 05:19 AM
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Default Re: Problem with Transgenic Mice

I have genotyped the tail for all mice. I have previously tried to create a knock in mouse, but was unsuccessful. I thought that microinjecting the foreign DNA would be a faster method.

I dont have a specific reason for believing that the integration occurred in the telomeres. If contamination is considered a plausible reasoning, I was thinking that integration in the telomere region is as valid and plausible reasoning. It doesnt make sense that when i first genotyped them, they showed up as positive, but when i genotyped them 3 months later, they were negative. I seriously doubt that they are mosaics, because the results should be consistent even if mosaic or not.
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Old 11-23-2011, 07:33 PM
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Default Re: Problem with Transgenic Mice

It sounds like the PCR assay may be flaky, it's probably best to check for the transgene by Southern blotting of your tail DNA.

In terms of time, pronuclear transgenesis can be faster, but the main problem is you have to generate multiple independent lines and the expression is not always stable over generations and the pattern of expression is heavily influenced by the integration site (do you have an IRES-GFP or IRES-lacZ built into your construct so you can check the pattern relatively easily?).

I think that if you want to generate a reagent that will be generally useful in the long term the best way to go is to do a knock-in. We have found that even BAC-transgenic mice often do not give the correct pattern of expression, so doing K-ins is the most sure fire way to get what you need.
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