I am trying to create stable cell lines and although I have positive clones the expression levels are very low. How could this be explained? In the past I used to have much higher efficiency in infection and some clones with high levels of expression. Could this be explained because of not many viruses infecting each cell and because the genome is incorporated fewer times in the genome?
As you say, it might be due to the number of virus copy inserted in your cells. In addition, it depend too if the insertion are in tandem or in inverted sens.
The insertion site in the genome is important too for the efficiency of your virus.
Are you titering your virus stock? Which MOI are you using?
It is the same transgene you are using in these new experiments? If you are using always the same MOI but with a different transgene and getting different results, maybe this one is toxic to the cells, thereby selecting only the clones with low copy.
We need more information...;)
you can also get high expression when only a single copy of the gene (the virus) is integrated. So it is not necessarily the low infection efficiency but the low expression levels could be also due to the genetic setup of the viral vector used.
Did you use the viral vector before and got better results? or is this a newly created vector?
Just in case.. are you using an LTR or CMV promotor driven expression system? ..and some promotors become silenced depending on the cell type (i.e. CMV in MEF cells)
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