Hi,
I am genotyping newly constructed trangenic mice knocking down target gene. I have got F1 generation from F0. And DNA examination indicates that the target DNA fragment has been excised. The bugs are that PCR targeting mRNA and WB show that the target mRNA and protein are not knocked down. PS: One of my mRNA primers locates in excised region. The only reason I think of is the PCR contamination and bad behavior of first antibody as DNA has been excised. Could you pls help me with this problems? THANK YOU.
Hi Jing,
For proper troubleshooting, I think more information is needed in your case. For example, how did you do your genotype validation (primer targets, etc) of the strain/progeny? Are you certain that the progeny that you are testing are homozygous for the knockdown?
You can check whether there is gDNA contamination by using a pair of primer that amplifies a region encompassing an intron (of maybe a housekeeping gene, such as the one that you use for qPCR control), so that you can tell based on the size of the amplicon whether it is from gDNA or mRNA.
Best,
Sheng
Run a full set of controls with your experiment. It's entirely possible that your mice do not have the transgenic knockdown at all. Better to learn that now with PCR than to waste months or years using a non-useful mouse line.
