Hi,
I am generating stable cell lines (HEK293A) with my gene of interest using the AAVS1 integration system. The integration site is present on Chromosome 13, which is triploid in HEK cells. I want to confirm how many copies of my gene of interest are present in the different clones I have got for my stable cell lines. One way of doing so is doing a qPCR on genomic DNA isolated from these cells but what control should I be using to estimate the copy number?
Use a housekeeping gene like tubulin which should be diploid and the SV40 large T antigen which should be haploid.
Also use PCR amplification products of your primers at specific concentrations to generate a calibration curve incase there are any differences in amplification efficiency between your primer pairs.
Hi Robert, thanks for your response. I was wondering would've tubulin have 3 copies as well because it is on Chr 12 which is triploid. Also, i am using HEK293A cells so they dont express the SV40 T antigen.
* also just an edit to my original question, the AAVS1 site is on Chr 19 (triploid).
Is actin an option? Even if the SV40 T antigen is not expressed, as long as it is not deleted you can amplify it.
Hi Robert,
SV40 T antigen is not present in HEK293A cells and is exclusive to HEK293T cell line. I have decided to go ahead with plotting a standard curve of ct value vs. copy number using the original plasmids I used to create these recombinant cell lines. This will still be relative but would ideally give me three different populations corresponding to different copy numbers of my gene of interest. What are your thoughts on this?
Possible but you would then need to calculate the number of plasmids per mass of plasmid DNA.
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