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Estimating copy number in transgenic mice

Tom Thatcher ttha at uhura.cc.rochester.edu
Thu Dec 21 13:03:09 EST 1995


In <8176.199512211527 at lenzie.cent.gla.ac.uk> gvsa07 at UDCF.GLA.AC.UK ("T.J.Anderson") writes:

>I would like to see what the opinion is on the best techniques
>available  to distinguish between hemizygous and homozygous mice with
>with low (3-7) copies of a transgene that contains a wild type
>gene. How confident would people be that they can distinguish
>between hemizygous and homozygous animals.
>The current probe hybridises to both transgene and wild type 
>DNA sequences.

>As a supplementry question I wonder what degree of confidence
>one might be able to count the extra numbers of the gene 
>contributed by the transgene and what is considered to be
>the "gold standard" technique for doing this.

The "gold standard" is to breed the mouse and check the offspring.
You can do dilutions of mouse and plasmid DNA on a Southern or dot
blot, and be reasonably sure of the difference between 2 copies, 
5-8 copies, and 8-16 copies (2, 2+x, 2+2x) but you should not then
breed the mice willy-nilly assuming all the offspring are transgenic
until you have checked a couple of litters (prefereably against
a known non-transgenic) and found no non-transgenic F1s.

-- 
Tom Thatcher                          | You can give a PC to a Homo habilis,
University of Rochester Cancer Center | and he'll use it, but he'll use it
ttha at uhura.cc.rochester.edu           | to crack nuts.



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