Linear amplification mediated (LAM)-PCR analysis reveals the presence of 5 vector integration sites in individually picked hematopoietic clones from mouse 7.23.

Multiple integration sites were detected in each of CFU-GM colonies analyzed, demonstrating the ability of lentiviral vectors to transduce multiple copies of the vector into human repopulating cells. Figure shows spredex gel band analysis following LAM-PCR on DNA from bone marrow and individually picked CFU-GM colonies from mouse 7.23. Similar band profiles could be seen in both the total bone marrow and colony analyses. Bone marrow and colony numbers 42, 55, and 56 are shown. All 5 integration bands are labeled, along with the internal control band, which is a byproduct of the reaction. Due to the sensitivity of the LAM-PCR technique, additional bands were occasionally amplified. Sequencing revealed that these additional bands contained identical sequences to the known integration sequences. However, their difference in size compared to the true integration site band was due to DNA fragment ligation prior to amplification, from the linker cassette ligation procedure. (Integration bands containing either an extra linker cassette or an extra linker cassette along with an additional fragment of human genomic DNA were seen.) All true integration bands contain the 116 base pair (bp) of vector sequence upstream of the vector/human genome junction. Bands < 116 bp were demonstrated to be the result of a recombination between the HIV LTR and the linker cassette and found not to contain human genomic DNA. These results conclusively demonstrate the ability of lentiviral vectors to transduce multiple vector copies into human repopulating cells.