Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures

OBJECTIVE: We wanted to determine whether the long-term bone marrow culture (LTBMC) transduction system would lead to efficient gene transfer and engraftment of murine repopulating hematopoietic stem cells (HSC), particularly in nonablated recipients. MATERIALS AND METHODS: Congenic mouse strains expressing Ly 5.1 or Ly 5.2 and the GP+E86 cell line producing the MGirL22Y vector carrying the gene for enhanced GFP were used. Murine LTBMCs were established and demi-depopulated on days 7 and 14 with addition of vector supernatant on days 8 and 15. RESULTS: Cell recovery on day 21 was 21.3%+/-3.8% of input cells and CFU-C recovery was 9.7+/-3.4% as compared with CFU-C of input cells. In vitro transduction efficiency determined by CFU-C expressing GFP was 22.2%+/-1.6%. In irradiated (950 cGy) mice transplanted with 2x10(6) LTBMC cells, 94% of nucleated cells in the blood at week 16 were of donor origin. However, GFP was only detected at low level in a few animals at week 4 and not later. Analysis of bone marrow from these mice at week 20 did not show any GFP expression and semiquantitative PCR revealed a transgene level of