Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells.
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.
Muramatsu
M
M
Division of Genetic Therapeutics, Center for Molecular Medicine, Tochigi, Japan.
Hanazono
Y
Y
Ogasawara
Y
Y
Okada
T
T
Mizukami
H
H
Kume
A
A
Mizoguchi
H
H
Ozawa
K
K
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
D018168:Receptors, Retinoic Acid; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases