title: Regulated expression of an MHC class II gene from a promoter-inducible retrovirus.
abstract: Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.
authors.affiliation: Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Sonntag
authors.forename: G W
authors.initials: GW
authors.lastname: Haller
authors.forename: D
authors.initials: D
authors.lastname: Giauffret
authors.forename: S
authors.initials: S
authors.lastname: Germana
authors.forename: S A
authors.initials: SA
authors.lastname: Reeves
authors.forename: J
authors.initials: J
authors.lastname: Levy
authors.forename: D H
authors.initials: DH
authors.lastname: Sachs
authors.forename: C
authors.initials: C
authors.lastname: LeGuern
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015152:Blotting, Northern; D015139:Blotting, Southern; D016026:Bone Marrow Transplantation / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D004727:Endothelium / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005802:Genes, MHC Class II / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics*; D013552:Swine; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014162:Transfection; D014184:Transplantation, Homologous / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D013752:Tetracycline; D004318:Doxycycline