Expression of a swine class II gene in murine bone marrow hematopoietic cells by retroviral-mediated gene transfer.
As a first step in assessing the efficacy of a gene transfer approach to the induction of transplantation tolerance in our miniature swine model, double-copy retroviral vectors engineered to express a drug-resistance marker (neomycin) and a swine class II DRB cDNA were constructed. Infectious particles containing these vectors were produced at a titer of greater than 1 x 10(6) G418-resistant colony-forming units/ml using both ecotropic and amphotropic packaging cell lines. Flow cytometric analysis of DRA-transfected murine fibroblasts subsequently transduced with virus-containing supernatants demonstrated that the transferred sequences were sufficient to produce DR surface expression. Cocultivation of murine bone marrow with high-titer producer lines leads to the transduction of 40% of granulocyte/macrophage colony-forming units (CFU-GM) as determined by the frequency of colony formation under G418 selection. After nearly 5 weeks in long-term bone marrow culture, virus-exposed marrow still contained G418-resistant CFU-GM at a frequency of 25%. In addition, virtually all of the transduced and selected colonies contained DRB-specific transcripts. These results suggest that a significant proportion of very primitive myelopoietic precursor cells can be transduced with the DRB recombinant vector and that vector sequences are expressed in the differentiated progeny of these cells.
Shafer
G E
GE
Transplantation Biology Research Center, Massachusetts General Hospital, Charlestown 02129.
Emery
D W
DW
Gustafsson
K
K
Germana
S
S
Anderson
W F
WF
Sachs
D H
DH
LeGuern
C
C
D000818:Animals; D001854:Bone Marrow Cells; D003001:Cloning, Molecular; D015870:Gene Expression; D005802:Genes, MHC Class II; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D013552:Swine; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger