Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development.
Unlike conventional gammaretroviral vectors, the murine stem cell virus (MSCV) can efficiently express transgenes in undifferentiated embryonic stem cells (ESCs). However, a dramatic extinction of expression is observed when ESCs are subjected to in vitro hematopoietic differentiation. Here we report the construction of a self-inactivating vector from MSCV, MSinSB, which transmits an intron embedded within the internal transgene cassette to transduced cells. The internal transgene transcriptional unit in MSinSB comprises the composite cytomegalovirus immediate early enhancer-chicken beta-actin promoter and associated 5' splice site positioned upstream of the natural 3' splice site of the gammaretroviral envelope gene, and is configured such that the transgene translational initiation sequence is coincident with the envelope ATG. MSinSB could be produced at titers approaching 10(6) transducing units/ml and directed higher levels of transgene expression in ESCs than a splicing-optimized MSCV-derived vector, MSGV1. Moreover, when transduced ESCs were differentiated into hematopoietic cells in vitro, MSinSB remained transcriptionally active in greater than 90% of the cells, whereas MSGV1 expression was almost completely shut off. Persistent high-level expression of the MSinSB gammaretroviral vector was also demonstrated in murine bone marrow transplant recipients and following in vitro myelomonocytic differentiation of human CD34(+) cord blood stem/progenitor cells.
Ramezani
Ali
A
Department of Anatomy and Cell Biology, The George Washington University Medical Center, Washington, DC, USA.
Hawley
Teresa S
TS
Hawley
Robert G
RG
D000818:Animals; D016026:Bone Marrow Transplantation; D051927:CD24 Antigen; D045744:Cell Line, Tumor; D002645:Chickens; D003587:Cytomegalovirus / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D004625:Embryo, Nonmammalian; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D017988:Gammaretrovirus / Q000235:genetics*; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007438:Introns; D007963:Leukocytes, Mononuclear; D051379:Mice; D022821:RNA Splice Sites; D013234:Stem Cells / Q000166:cytology*; D020079:Terminal Repeat Sequences / Q000235:genetics; D014158:Transcription, Genetic
D051927:CD24 Antigen; D022821:RNA Splice Sites; D005914:Globins