High-frequency cell surface expression of a foreign protein in murine hematopoietic stem cells using a new retroviral vector.
A retroviral vector (pSFF) derived from murine Friend spleen focus forming virus was used to transduce murine hematopoietic stem cells and express a cell surface marker protein, mutated murine prion protein, in vitro and in vivo after transplantation. To enhance retroviral vector integration in bone marrow cells, mice were treated with 5-fluorouracil (5-FU) to increase stem cell mitotic activity, which peaked on day 8 post-5-FU. The infectivity titer of the vector, pSFF-mPrP-3F4, was determined by a novel assay in which antigen-positive foci of infected cells were detected after replication and spread of the vector in cultures of mixed packaging cell lines. Infection of Sca-1+/Lineageneg-low cells with pSFF-mPrP-3F4 resulted in marker protein expression in 40% of the progeny cells after 7 days of culture. Transplantation of marrow cells or sorted Sca-1+/Lineageneg-low cells transduced with vector resulted in 3F4-positive mPrP expression in 11% to 37% of donor-derived peripheral blood leukocytes at 2 weeks. Though the percentage of 3F4-positive blood cells gradually declined, at 28 weeks 23% of recipient mice still maintained expression of the marker gene. Expression was observed in lymphoid, myeloid, and erythroid lineages and was detected in Sca-1+/Lineageneg-low marrow cells. The multilineage, high-frequency expression observed suggests that pSFF may be useful in gene therapy directed at hematopoietic stem cells and their differentiated progeny.
Tumas
D B
DB
Laboratories of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, MT 59840, USA.
Spangrude
G J
GJ
Brooks
D M
DM
Williams
C D
CD
Chesebro
B
B
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D019070:Cell Lineage; D003673:Defective Viruses / Q000235:genetics*; D005472:Fluorouracil / Q000494:pharmacology; D005622:Friend murine leukemia virus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000821:virology; D016130:Immunophenotyping; D051379:Mice; D008810:Mice, Inbred C57BL; D018621:PrPC Proteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012091:Repetitive Sequences, Nucleic Acid; D013155:Spleen Focus-Forming Viruses / Q000235:genetics*; D014162:Transfection
D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D005472:Fluorouracil