Copy number of Transgene in Trangenic Mice

Bryan J. Maloney cavaggione at sbcglobal.nmungemungt
Tue Feb 3 22:59:41 EST 2004


"Obaid Khan" <oyk01482 at creighton.edu> nattered on
thusnews:124b8d458d265f1b9c27ee4691e3b695 at news.teranews.com: 

> I will appreciate if anyone can guide me how to calculate the copy
> number of transgenes in mice using southern blot. I have come across
> some methods on the net and that are sort of conflicting. I will
> appreciate if anyone who has done this will give me some tips and
> directions on how to spike the genomic DNA with the transgene and the
> calculations involved. 

There are a couple of ways to do it.  One is densitometric and can be a 
pain in the patoot--a MAJOR pain in the patoot.  

The other is a bit more old-fashioned and elegant.

Select some enzymes that cut only once in the insert--near the end of the 
insert (including flanking sequences that are inserted).  Cut a couple of 
samples with each enzyme (separately).  Use probes that span the enzyme 
sites (you will need to make multiple probes for this).  You should get 
twice as many bands lighting up as you have copies of the transgene.  The 
worst-case scenario of managing to have adventitious sites on either side 
of the transgene spaced to produce products very close in size is highly 
unlikely.  The only problem is if the adventitious site(s) produce very 
large DNA fragments--still unlikely if you use a specific six-cutter.




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