Variable transgene copy number? - Transgene copy number varies in rats :( (Mar/20/2006 )
I am trying to genotype transgenic rats using real-time PCR. These rat were produced more than a decade ago using microinjection with random insertion of the transgene.
It looks like these rats have wide range of variability in the copy number of the transgene. Instead of having just three type: homozygotes with 2X copies, heterozygots with 1X and wild-types with 0 copies of the gene, I observed that there are some animals with for example, 3X, 5.5X sometimes 0.4X copy number of the transgene. We have repeated the PCR many times and we still observe these strange ratios. (In our case the copy number for a "heterozygote" equals 30-32, so homozygotes would be around 60, and even though 'heterozygote" and "homozygote" animals are more common, some animals seem to have 45, 85, sometimes 20 copies of the transgene.)
My question is: is it possible that the transgene randomly inserted itself into several different chromosomes and now we see the result of chromosomes with different copy number of the transgene being randomly distributed in meiosis producing these various "in-between" transgene copy numbers?
You might be interested in these articles (they're about plants, but it's the same principle)
Song P, Cai CQ, Skokut M, Kosegi BD, Petolino JF. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS -derived transgenic maize. Plant Cell Reports. 2002;20:948–954.
Mason G, Provero P, Varia AM, Acotto GP. Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnology. 2003;2:20.
They talk about yield the high proportion of transformants with highly differing numbers of transgenes and only a small proportion of primary transformants with low copy numbers, which was picked up by Real Time, and not Southerns.
Ballester M, Castello A, Ibanez E, Sanchez A, Folch JM. Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals.
Biotechniques. 2004 Oct;37(4):610-3.
This paper seems like it migh be able to help, but I don't have access to it (perhaps you do).
From what I can recall, there are cases when the transgene is present in a number of chromosomes (which can cause problems when it is in a locus where it is silenced, or it interacts with .... things). Sorry, I can't give you a definitive answer.