Can I still use PCR to select transfectatns - (Nov/25/2008 )

Hi everyone,

I am really confused. I transfected the pGL3-control vector (without any other insert) into a cancer cell line together with Hygromycin B resistance containing vector. The cells have selected in the selective medium for a while. I didnot linearize the vectors when I transfected the cells. Do you think I still can extract the cells gonome and do PCR to select for colonies having luciferase? Also, is it possible that the luciferase gets integrated but the SV40 does not and gets missing during transfection? If I still can use PCR, can I design primers that give a product including both SV40 and luciferase? Thanks you so much for reading this and any suggestion!!

Oh, happy coming thanksgiving!!

Yang

I'm guessing here, but do you have hygro-resistant cells that do not appear to make luciferase? I had this problem a while ago, and took some time to figure out how to tackle it. Hopefully this will help.....

It seems to me that you would be better off to have put both your luciferase and hygromycin genes into the same vector. If they are separate, you have no real confidence that both vectors were integrated. Initially, you'd have at least 4 types of colonies, (1) non-transfected, (2) Hygro only, (3) Luc only and (4) Hygro + Luc. After selection, you should have only colonies 2 and 4 remaining, but cannot really differentiate between the two. Further, if the Luc and Hygro are integrated into two, relatively distant different places in the genome, you may have cells that have both genes present in the genome on PCR, but due to methylation or other silencing mechanism, you don't express Luc, even though the gene and constitutuively active promoter are 'present'.

If you cannot make a new vector, I'd recommend validating your colonies first by RT-PCR to detect the transcript, and then a luminescence reading to be sure you've got cells that make the protein as well.

As for your question about integrating the Luc gene but not SV40, I suppose it is possible though not particularly likely, as they are rather close on the pGL3-control vector. Relative to the whole genome this stretch of DNA (~2.4kb) is but a speck.

And yes, you can design primers to amplify both.