Fibroblast transplantation in rats: transduction and function of foreign genes.
The purpose of this study was to develop a method of transduction of normal skin fibroblasts with a retroviral vector expressing the human factor IX (hF-IX) gene and transplantation of the transduced cells into rats. A retroviral vector containing the hF-IX gene, as well as a selectable marker (hygromycin B resistance; HB), was used to transduce cultured normal skin (rat and human) fibroblasts. Transduced fibroblasts were selected with HB; resistant clones were propagated and assessed for expression of active hF-IX in vitro. Transduced cells secreted significant amounts of hF-IX in vitro (327.9 +/- 14.2 ng/10(6) cells) which showed measurable clotting activity, indicating terminal processing of the gene-transcribed hF-IX product by the transduced fibroblasts. Integration of the proviral gene into rat and human fibroblast genomic DNA was confirmed by polymerase chain reaction. Transduced rat fibroblasts were attached to collagen-coated microcarriers and transplanted intraperitoneally into syngeneic recipients. Transplanted microcarrier-attached cells formed aggregates in the peritoneal cavity and exhibited positive immunohistochemical staining for hF-IX up to 8 weeks following transplantation, the time of termination of the experiment. These experimental results may have significant implications in developing strategies for future clinical therapy for hemophilia B.
Neuzil
D
D
Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee 37203.
Holzman
M
M
Rozga
J
J
Levine
J
J
Moscioni
A D
AD
Demetriou
A A
AA
D000818:Animals; D017690:Cell Transplantation; D005164:Factor IX / Q000235:genetics*; D005347:Fibroblasts / Q000637:transplantation*; D005822:Genetic Vectors; D006801:Humans; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D014162:Transfection
D005164:Factor IX