SF3B1 KNOCKDOWN AT EARLY ERYTHROPOIESIS INDUCED CELL APOPTOSIS BY USING A TET-ON LENTIVIRAL VECTOR
Author(s): ,
Yumin Huang
Affiliations:
Department of Hematology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou,China
,
Xianfang Wu
Affiliations:
Laboratory of Virology and Infectious Disease,the Rockefeller University,New York,United States
,
Ling Sun
Affiliations:
Department of Hematology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou,China
Xiuli An
Affiliations:
Laboratory of Membrane Biology,New York Blood Center,New York,United States
EHA Library. Huang Y. Jun 15, 2019; 266845; PS1228
Yumin Huang
Yumin Huang
Contributions
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Abstract

Abstract: PS1228

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
SF3B1 has been drawing great interests of scientists due to their significantly high mutation frequency in one kind of hematological disease Myelodysplastic syndromes-Refractory anemia with ring sideroblasts (MDS-RARS), which is characterized by the anemia in clinic. However, the physiological roles of SF3B1 in erythropoiesis are poorly studied.

Aims
To elucidate the biological functions of SF3B1 in specific stages of human erythropoiesis through precise temporal control of SF3B1 expression. To test the efficiency and safety of the new TET-on lentiviral vector in differentiating cells.

Methods
TET-on lentiviral vector was constructed for conditional gene knockdown. Undifferentiated CD34+ cells were transduced with the Lentivirus and SF3B1 gene knockdown was induced by doxycycline in different differentiation stages across human erythropoiesis. The efficiency and safety of the TET-on lentiviral vector were assessed by monitoring green fluorescent protein expression and erythroblasts differentiation, respectively. Knockdown of SF3B1 gene was checked by using qRT-PCR and western blot analyses. The effects of SF3B1 ablation on progenitors were detected by colony forming assay and apoptosis was assessed by flow cytometry.

Results
The TET-on vector is efficient and safe for erythroblasts with no disturbance to the proliferation, differentiation, morphology and enucleation. Knockdown of SF3B1 in erythroblasts impairs erythroid colony forming and induced cell apoptosis. Interestingly, knockdown of SF3B1 shows temporal effects on erythropoiesis: in early stages, the deficiency of SF3B1 leads to significant cell apoptosis; however, it has no effect on cell growth at late stages of erythropoiesis.

Conclusion
Our findings indicate that the TET-on lentiviral vector is a powerful tool for precise temporal control of gene expression in differentiating cells, and SF3B1 is critical for the growth of early erythropoiesis.  

Session topic: 23. Hematopoiesis, stem cells and microenvironment

Keyword(s): Erythropoieisis, MDS

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