| Co-Investigator(Kenkyū-buntansha) |
SAKATA Tsuneaki DNAVEC Research Inc., Senior Scientist (1997), (研究職)室長
HASEGAWA Mamoru DNAVEC Research Inc., Director, (研究職)所長
URABE Masashi Jichi Med.Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (40213516)
KUME Akihiro Jichi Med.Sch., Faculty of Medicine, Assistant Professor, 医学部, 講師 (10264293)
|
|---|
| Research Abstract |
To overcome the low efficiency of gene transfer into hematopoietic stem cells, we have developed a novel strategy for selective expansion of transduced hematopoietic cells. A fusion gene encoding a chimeric receptor (DELTAGCRER) between G-CSF receptor (G-CSFR) and hormone-binding domain (HBD) of estrogen receptor (ER) was constructed as a prototype of "selective amplifier genes (SAG)". G-CSFR was employed as a signal generator and the G-CSF-binding domain was deleted not to respond to G-CSF.ER-HBD was employed as a molecular switch to control the activity of fusion protein in estrogen (E_2)-dependent manner. Although E_2-inducible growth was achieved when BaIF3 cells were transduced with this SAG, it also induced differentiation in transduced 32D cells upon B2 treatment. Since only a growth signal is required, we modified the DELTAGCRER gene to attenuate the differentiation signal. Phe was substituted for Tyr7O3 in the chimeric receptor. When the resultant SAG (DELTAY7O3F-GCRER gene) was expressed in 32D, sustained growth was observed with minimal differentiation upon E_2 treatment. The findings suggest that DELTAY7O3F-GCRBR gene can function as a more desirable SAG.In addition, a mutant ER (TmR), which specifically binds to a synthetic ligand 4-hydroxytamoxifen (Tm), was used as another molecular switch. When GCRTmR was expressed in Ba/F3 cells, Tm-dependent growth was observed with little response to E_2. The GCRTmR/Tm system may be valuable when the SAG method is applied to in vivo amplification of transduced hematopoietic cells, because the influences of endogenous E_2 can be eliminated.
|
