genotyping transgenic mice with PCR - (Feb/23/2005 )
I know the methods for detecting heterozygous transgenic mice using PCR. But want know how to discriminate homozygous from heterozygous transgenic mice. Is it possible to do it with conventional PCR? or Real-time PCR is required? How should primers be designed if the transgene integration site is not known?
If anyone out there can help, I would appreciate it very much. Thank you.
I have used a system using three primers to distinguish hetero from homozygous mice. In this case we had: common primer, wild type primer and ko primer. Putting all three in the PCR enabled us to tell the genotype of the mouse as there were two band sizes i.e. 1 big band = homozygous +/+, two bands = +/-, and 1 little band= -/-.
Sorry can't help you with the primers, but perhaps you could try labelling a primer for the sequence you know, then adding a random primer (e.g. RAPD primer) and sequencing any bands that show up as labelled, hopefully you will get some sequence outside that which you already know.
Best of luck