New selective amplifier genes containing c-Mpl for hematopoietic cell expansion.
We previously developed "selective amplifier genes (SAGs)" which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34(+) cells than the GCR-type SAG. One mutant Mpl-type SAG (Delta GCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.
Nagashima
Takeyuki
T
DNAVEC Research, Incorporated, 1-25-11, Kannondai, Tsukuba-shi, Ibaraki-Ken 305-0856, Japan.
Ueda
Yasuji
Y
Hanazono
Yutaka
Y
Kume
Akihiro
A
Shibata
Hiroaki
H
Ageyama
Naohide
N
Terao
Keiji
K
Ozawa
Keiya
K
Hasegawa
Mamoru
M
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D001665:Binding Sites; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D017353:Gene Deletion; D005821:Genetic Techniques; D015316:Genetic Therapy; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000235:genetics* / Q000378:metabolism*; D053628:Receptors, Thrombopoietin; D012190:Retroviridae / Q000235:genetics
D018952:Antigens, CD34; D007377:Interleukin-3; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human