Development of a self-inactivating tet-on retroviral vector expressing bone morphogenetic protein 4 to achieve regulated bone formation.
The aims of this study were to explore the possibility of improving the design of self-inactivating (SI) retroviral vectors and to develop an SI vector that would allow optimal tet-on-regulated therapeutic gene expression. To minimize any interference between the viral promoter and the inducible promoter, we deleted different regulatory elements in the 3'LTR and examined their effects on transgene expression in transfected or transduced cells. In transfected cells, such deletions reduced the transgene expression. The insertion of a polyadenylation sequence could not completely compensate for this effect. We observed three patterns of transgene expression in cells transduced with these tet-on retroviral vectors: (1) high levels of both basal and inducible expression, (2) low levels of both basal and inducible expression, and (3) low levels of basal and high levels of inducible expression. After using the optimal vector to transduce muscle-derived stem cells, we were able to regulate the strong in vitro expression of transgenes-including enhanced green fluorescent protein and bone morphogenetic protein 4-via the addition or withdrawal of doxycycline (Dox). Implantation of the transduced cells and subsequent Dox-dependent induction of gene expression resulted in bone formation in vivo. Thus, we have developed an optimal SI retroviral vector that maintains a high titer, efficiently transduces muscle-derived stem cells, and enables both high levels of inducible gene expression in vitro and robust regulated bone formation in vivo.
Peng
Hairong
H
Growth and Development Laboratory, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA.
Usas
Arvydas
A
Gearhart
Brian
B
Young
Brett
B
Olshanski
Anne
A
Huard
Johnny
J
D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000032:analysis / Q000235:genetics*; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D032446:Myoblasts / Q000737:chemistry; D010012:Osteogenesis; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D016385:TATA Box / Q000235:genetics; D020079:Terminal Repeat Sequences / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004318:Doxycycline