A novel human artificial chromosome vector provides effective cell lineage-specific transgene expression in human mesenchymal stem cells.
Mesenchymal stem cells (MSCs) hold promise for use in adult stem cell-mediated gene therapy. One of the major aims of stem cell-mediated gene therapy is to develop vectors that will allow appropriate levels of expression of therapeutic genes along differentiation under physiological regulation of the specialized cells. Human artificial chromosomes (HACs) are stably maintained as independent chromosomes in host cells and should be free from potential insertional mutagenesis problems of conventional transgenes. Therefore, HACs have been proposed as alternative implements to cell-mediated gene therapy. Previously, we constructed a novel HAC, termed 21 Deltapq HAC, with a loxP site in which circular DNA can be reproducibly inserted by the Cre/loxP system. We here assessed the feasibility of lineage-specific transgene expression by the 21Deltapq HAC vector using an in vitro differentiation system with an MSC cell line, hiMSCs, which has potential for osteogenic, chondrogenic, and adipogenic differentiation. An enhanced green fluorescent protein (EGFP) gene driven by a promoter for osteogenic lineage-specific osteopontin (OPN) gene was inserted onto the 21 Deltapq HAC and then transferred into hiMSC. The expression cassette was flanked by the chicken HS4 insulators to block promoter interference from adjacent drug-resistant genes. The EGFP gene was specifically expressed in the hiMSC that differentiated into osteocytes in coordination with the transcription of endogenous OPN gene but was not expressed after adipogenic differentiation induction or in noninduction culture. These results suggest that use of the HAC vector is suitable for regulated expression of transgenes in stem cell-mediated gene therapy.
Ren
Xianying
X
Department of Molecular and Cell Genetics, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori 683-8503, Japan.
Katoh
Motonobu
M
Hoshiya
Hidetoshi
H
Kurimasa
Akihiro
A
Inoue
Toshiaki
T
Ayabe
Fumiaki
F
Shibata
Kotaro
K
Toguchida
Junya
J
Oshimura
Mitsuo
M
D000971:Antineoplastic Combined Chemotherapy Protocols; D002454:Cell Differentiation; D019070:Cell Lineage; D022222:Chromosomes, Artificial, Human / Q000235:genetics* / Q000378:metabolism*; D003520:Cyclophosphamide; D004317:Doxorubicin; D017353:Gene Deletion; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D053495:Osteopontin; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D014750:Vincristine
C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C485184:enhanced green fluorescent protein; D053495:Osteopontin; D049452:Green Fluorescent Proteins; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide