A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells.
BACKGROUND
We have developed a novel system for expansion of gene-modified hematopoietic stem/progenitor cells to overcome the low efficiency of current gene transfer methodology. This system involves 'selective amplifier genes', that encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCR) and the hormone-binding domain of estrogen receptor (ER). Hematopoietic progenitors expressing the chimeras showed estrogen-responsive growth in a controllable manner. However, endogenous estrogen may activate the fusion proteins in vivo, depending on the hormonal status of the subjects.


METHODS
We replaced ER with a mutant receptor (TmR) which specifically binds to 4-hydroxytamoxifen (Tm), to overcome limitations with wild-type ER. Interleukin-3 (IL-3)-dependent Ba/F3 cells and hematopoietic progenitor cells transduced with the resultant fusion proteins (GCRTmR and delta GCRTmR) were examined for ligand-inducible growth.


RESULTS
GCRTmR- and delta GCRTmR-expressing Ba/F3 showed IL-3-independent growth in response to Tm, while the cells were unresponsive to estrogen at concentrations up to 10(-7)-10(-6) M. Furthermore, murine bone marrow cells transduced with GCRTmR and delta GCRTmR formed colonies in methyl-cellulose medium in response to Tm, while virtually no colonies appeared with 10(-7) M estrogen or without cytokines.


CONCLUSIONS
These results suggest that influences of the endogenous estrogen can be almost eliminated by using the GCRTmR/Tm or delta GCRTmR/Tm system to expand gene-modified hematopoietic stem/progenitor cells.
Xu
R
R
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan.
Kume
A
A
Matsuda
K M
KM
Ueda
Y
Y
Kodaira
H
H
Ogasawara
Y
Y
Urabe
M
M
Kato
I
I
Hasegawa
M
M
Ozawa
K
K
D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000235:genetics; D004958:Estradiol / Q000494:pharmacology; D005784:Gene Amplification; D015316:Genetic Therapy; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013629:Tamoxifen / Q000494:pharmacology*; D014161:Transduction, Genetic
D017931:DNA Primers; D011960:Receptors, Estrogen; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; D004958:Estradiol