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 believe d to be a fast and convenient method for this purpose when using primers fl anking the cloning site of the inserted DNA. Unfortunately, a single PCR re action often fails to amplify the targeted fragment because of the existenc e of endogenous virus DNA in cell genome. However, in this study we conduct ed 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 retr oviral vectors in 22 transfected cell lines. We also proved that real time quantitative PCR in combination with MFG-specific primer is useful to deter mine copy number of the retroviral vector in murine producer cell lines.