F36VFGFR1 supports the survival of long-term repopulating HSCs during growth factor deprivation. Marrow cells (5 × 105) transduced with the F36VFGFR1 vector were transplanted into lethally irradiated recipients either immediately following transduction (A) or after 1 (B) or 5 days (C) of culture in the presence (solid symbols) or absence (open symbols) of 100 nM AP20187. Mice that were given transplants of cultured cells received all the progeny generated in cultures initiated with 5 × 105 transduced cells at day 0. Each line depicts results from a single mouse. One mouse (6281) that was given transplants of cells cultured for 5 days in AP20187 was killed at day 174 after transplantation and 5 × 106 marrow cells from this mouse were transplanted into 2 lethally irradiated secondary recipients (symbols × and +). E indicates erythroid; P, platelets, G, granulocytes; M, monocytes; B, B cells; and T, T cells. (C; inserts) LAM-PCR using RsaI confirms common provirus insertion patterns in the granulocytes (G), monocytes (M), B cells (B), and T cells (T) of mouse 6281. MW indicates DNA ladder. Similar results were obtained using 2 other restriction enzymes, Tsp509 I and HaeIII. Sequencing of LAM-PCR products from mouse 6281 (panel D) revealed provirus insertion sites at the indicated positions in chromosomes 4 and 16, and their presence in the indicated lineages was confirmed by PCR using using an LTR primer, a host genomic primer, and 38 cycles of amplification. A second independent experiment performed in serum-free conditions showed a similar trend at 64 days after transplantation.
