A speedy method to detect inserted DNA fragment in cell lines transfected with retroviral vectors

Cells transfected by retroviral vectors are brought in a gene of particular interest and are very useful in a variety of experiments. It is essential to testify that the DNA fragment was successfully introduced into the cells together with the retroviral vectors. Polymerase chain reaction is believed to be a fast and convenient method for this purpose when using primers flanking the cloning site of the inserted DNA. Unfortunately, a single PCR reaction often fails to amplify the targeted fragment because of the existence of endogenous virus DNA in cell genome. However, in this study we conducted a procedure for a single PCR, using vector-specific primers as well as a nested PCR, and successfully detected the DNA fragments cloned in MFG retroviral vectors in 22 transfected cell lines. We also proved that real time quantitative PCR in combination with MFG-specific primer is useful to determine copy number of the retroviral vector in murine producer cell lines.