title: Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells.
abstract: Since hematopoietic stem cells (HSCs) differentiate readily ex vivo resulting in the loss of self-renewal and engraftment abilities, the transient block of differentiation is essential to maintain those abilities during their ex vivo expansion culture. To this end, we developed a method of reversible integration of the dominant negative retinoic acid receptor (DN-RAR) gene, a differentiation-blocking gene, into cells utilizing the Cre/loxP-dependent gene recombination system. The murine immature hematopoietic 32D cells differentiate into mature neutrophils upon G-CSF treatment. However, 32D cells transduced with a retroviral vector expressing the DN-RAR gene put between two loxP sites continued to proliferate without showing differentiation even in the presence of G-CSF. After the cells were fully amplified, the cells were transduced with the Cre recombinase gene. The cells then restored the ability to differentiate into mature neutrophils upon G-CSF treatment. PCR analysis showed that the DN-RAR gene was efficiently removed from the genome by introduction of the Cre gene. This system may eventually be applicable to the ex vivo expansion of HSCs.
authors.affiliation: Division of Genetic Therapeutics, Center for Molecular Medicine, Tochigi, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Muramatsu
authors.forename: Y
authors.initials: Y
authors.lastname: Hanazono
authors.forename: Y
authors.initials: Y
authors.lastname: Ogasawara
authors.forename: T
authors.initials: T
authors.lastname: Okada
authors.forename: H
authors.initials: H
authors.lastname: Mizukami
authors.forename: A
authors.initials: A
authors.lastname: Kume
authors.forename: H
authors.initials: H
authors.lastname: Mizoguchi
authors.forename: K
authors.initials: K
authors.lastname: Ozawa
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D019426:Integrases; D051379:Mice; D009504:Neutrophils / Q000166:cytology*; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014764:Viral Proteins
chemical_list: D018168:Receptors, Retinoic Acid; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases