copy number of the trasgenic mice?

[jenny06]

Hi forums:
I have some problem to test the trasgene in mice, I already get the junction site PcR positive, so I want to make sure whether it is true in southern blot, so a liitle confusing to do it. Anyone has experience to plan the stratedy to get the copy number? very happy to come this forum. Thanks.

[kiki06]

Hey Jenny!
welcome to the forum.

To assess copy number of the transgenic mice you could:

1) check gene copy numbers in transgenic mice by serial dilution curves obtained by real-time quantitative PCR assay

2) Determine gene copy number by competitive PCR

3) Southern blot is the standard for copy #

In southern blot analysis of copy number, the intensity of the hybridization signal will correspond to the copy number of the transgene in the insertion site.

For more info check here:
http://www.med.umich.edu/tamc/tgoutline.html

Cheers,
post here your response

[jenny06]

THANKS. Another problem is the transgene is very similar to endpgenous gene, for exampe, if I choose carefully one enzyme to cut only once in trangene, I can figure out the transgene size, my question is what is the size the endogenous gene (this enzyme cann't cut the endogenous gene), i am little confusing? very appreciate your help!

[admin]

Hey Jenny,
thats a difficult situation. Did you by any chance Tag the recombinant protein (transgene) by myc or other tag? This would make it quite easy to find.