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New selective amplifier genes containing c-Mpl for hematopoietic cell expansion
Takeyuki Nagashima1, Yasuji Ueda, Yutaka Hanazono
1DNAVEC Research, Incorporated, 1-25-11, Kannondai, Tsukuba-shi, Ibaraki-Ken 305-0856, Japan.
Biochemical and Biophysical Research Communications
|March 21, 2003
Summary
Modified selective amplifier genes (SAGs) using the thrombopoietin (TPO) receptor show enhanced cell proliferation. These TPO-receptor SAGs offer tamoxifen-dependent cell expansion, with a mutant version losing TPO responsiveness.
Area of Science:
- Molecular Biology
- Cell Biology
- Hematology
Background:
- Selective amplifier genes (SAGs) enhance transduced cell growth.
- Existing SAGs utilize the G-CSF receptor (GCR) and are activated by tamoxifen (Tm).
Purpose of the Study:
- To investigate modified SAGs incorporating the thrombopoietin (TPO) receptor (c-Mpl) for enhanced signaling.
- To develop TPO-receptor SAGs dependent on Tm but not endogenous TPO.
Main Methods:
- Retroviral transduction of Ba/F3 cells with Mpl-type SAGs.
- Construction and testing of mutant Mpl-type SAGs for TPO-insensitivity.
- Assessment of cell proliferation in Ba/F3 and CD34(+) cells.
Main Results:
- Mpl-type SAGs promoted Tm- and TPO-dependent Ba/F3 cell growth without IL-3.
- Mpl-type SAGs exhibited greater proliferation induction in Ba/F3 and CD34(+) cells compared to GCR-type SAGs.
- A mutant Mpl-type SAG (Delta GCRMplTmR) demonstrated loss of TPO responsiveness while maintaining Tm-dependence.
Conclusions:
- Mpl-type SAGs represent a more potent system for cell expansion than GCR-type SAGs.
- A TPO-insensitive, Tm-dependent Mpl-type SAG was successfully engineered.