Use of recombinatory PCR to insert subtle genetic markers into Moloney murine leukemia virus-based retroviral vectors

As tools to examine template switches and recombination events during the process of reverse transcription, two nearly identical Moloney murine leukemia virus-based (MoMLV) retroviral vectors were constructed using the technique of recombinatory polymerase chain reaction (PCR). The experimental vectors designed for this study were based on the well-characterized LN series vectors. The protein coding regions normally present in the retroviral genome have been replaced by the coding regions for two drug resistance markers, neomycin phosphotransferase (Neo) and hygromycin phosphotransferase (Hyg). With only one functional drug resistance gene in each vector, the individual vectors as well as recombination events between them can be followed by phenotypic selection. Utilization of recombinatory PCR allowed the insertion of very subtle nucleotide changes resulting in a series of restriction site polymorphisms in the two retroviral vectors. The ability to create these subtle mutations in specific locations of these retroviral vectors allowed the utilization of naturally occurring areas of variability in the vectors and avoid regions important for replication.