Retroviral vector-mediated gene expression in human CD34+CD38- cells expanded in vitro: cis elements of FMEV are superior to those of Mo-MuLV.
A novel murine stromal cell line, HESS-M28, was established, which supports the expansion of human CD34+CD38- cells more than 300-fold in vitro in the presence of human IL-3 and SCF. These cells were used in an attempt to evaluate cis-acting elements of retroviral vectors in human primitive hematopoietic cells. Cord blood cells were cultured on top of the mixed cell layers of the stromal cell line, HESS-M28, and retroviral vector-producing cells. The FMEV-type vector SF/Lyt contained the spleen focus-forming virus U3 and the MESV primer-binding site (PBS), while MO3/Lyt contained the U3 region and PBS from Mo-MuLV. After transduction by the FMEV-type and Mo-MuLV-based vectors, expression of the marker gene murine CD8 (mCD8) was examined in CD34-, CD34+, and CD34+CD38- cells. In CD34+ and CD34+CD38- cells, expression of mCD8 was higher with the FMEV-type vector, SF/Lyt, compared with the cells transduced by the Mo-MuLV-based vector MO3/Lyt, although the expression was comparable in CD34- cells. Expression of marker genes was also confirmed in long-term culture-initiating cells (LTC-ICs) and SCID-repopulating cells (SRCs).
Tsuji
T
T
Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Japan.
Itoh
K
K
Baum
C
C
Ohnishi
N
N
Tomiwa
K
K
Hirano
D
D
Nishimura-Morita
Y
Y
Ostertag
W
W
Fujita
J
J
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000235:genetics*; D000943:Antigens, Differentiation / Q000235:genetics*; D016827:CD8 Antigens / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D005312:Fetal Blood / Q000378:metabolism; D005434:Flow Cytometry; D005622:Friend murine leukemia virus / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping; D019868:Kanamycin Kinase / Q000235:genetics; D008562:Membrane Glycoproteins; D051379:Mice; D008809:Mice, Inbred C3H; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008908:Mink Cell Focus-Inducing Viruses / Q000235:genetics*; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009244:NAD+ Nucleosidase / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D016827:CD8 Antigens; D004279:DNA, Viral; D008562:Membrane Glycoproteins; D019868:Kanamycin Kinase; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1