Retrovirus-mediated transfer and expression of an allogeneic major histocompatibility complex class II DRB cDNA in swine bone marrow cultures.
The possibility of inducing transplantation tolerance by somatic gene transfer is under investigation in our miniature swine model. As a crucial step in this project, we have used a retroviral vector engineered to express both a drug-resistance gene (Neo) and a swine class II DRB cDNA to transduce porcine bone marrow (BM) cells. Analysis of cultured swine fibroblasts exposed to high-titer viral supernatants demonstrated that drug resistance had been conferred and that transferred vector sequences were transcribed appropriately. Similar transduction studies with swine BM demonstrated the transfer of drug resistance to as high as 14% of colony-forming unit-granulocyte-macrophage (CFU-GM). Using polymerase chain reaction (PCR) of cDNA, vector-derived allogeneic DRB transcripts were detected in colonies derived from primitive CFU-Mix and high proliferative potential-colony-forming cell (HPP-CFC), as well as in drug-resistant GM colonies grown from transduced bone marrow (BM) maintained in long-term BM cultures (LTBMCs) for up to 5 weeks. These results indicate that a significant proportion of both colony-forming progenitors and LTBMC-initiating cells were transduced with the DRB-recombinant retroviral vector and that both vector-derived genes were expressed in the differentiated progeny of these cells.
Emery
D W
DW
Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Charlestown 02129.
Shafer
G E
GE
Karson
E M
EM
Sachs
D H
DH
LeGuern
C
C
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001853:Bone Marrow / Q000502:physiology; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006684:HLA-DR Antigens / Q000096:biosynthesis* / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012190:Retroviridae / Q000235:genetics; D013552:Swine; D013556:Swine, Miniature; D014162:Transfection / Q000379:methods*
D006684:HLA-DR Antigens; D009838:Oligodeoxyribonucleotides; D004247:DNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase