Construction of small interference RNA lentiviral Vector for M3 muscarinic acetylcholine receptor gene of bladder smooth muscle cells

doi:10.3969/j.issn.1006-7795.2018.03.016

Abstract

Abstract: Objective To construct M₃ acetylcholine receptor gene interference RNA lentiviral vectors for bladder smooth muscle cells.Methods Four pairs of small interference RNA (siRNA) sequence for M₃ muscarinic acetylcholine receptor gene were synthesized to construct GV118-sh-M₃ lentivirus vectors. DNA sequencing was used to identify the sequence. GV118-sh-M₃ lentivirus vectors were cotransfected with pHelper 1.0 and pHelper 2.0 vectors into 293T cells. We harvested the viral particles and assayed the titer of lentivirus by fluorescence. BSMCs were primary cultured in vitro and transfected by lentivirus of four groups (KD1, KD2, KD 3 and KD4). Fluorescence expression was observed and the expression levels of M₃ gene in BSMCs of groups were assessed by RT-PCR and Western blotting was used to calculate interference efficiency.Results Four GV118-sh-M₃ lentivirus vectors were successfully constructed and the sequencing results were consistent with sequence of the design. Viral particles of groups were harvested with titer of 2×10⁸, 6×10⁸, 5×10⁸ and 5×10⁸ TU/mL, respectively. KD1 group was with the highest fluorescent expression and interference efficiency.Conclusion The GV118-sh-M₃ interference lentivirus vector was successfully constructed and selected, which may lay a foundation for further study on the study of M₃ muscarinic acetylcholine receptor gene interference in treating neurogenic bladder.

Key words: neurogenic bladder, M₃ muscarinic acetylcholine receptor, lentiviral vector, RNA interference

CLC Number:

R694⁺.5

Shang Zhenhua, Jia Chunsong, Yan Hao, Wang Jukun, Wang Xu, Cui Bo, Wang Qi, Ou Tongwen. Construction of small interference RNA lentiviral Vector for M₃ muscarinic acetylcholine receptor gene of bladder smooth muscle cells[J]. Journal of Capital Medical University, 2018, 39(3): 398-403.

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URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2018.03.016

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2018/V39/I3/398

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Construction of small interference RNA lentiviral Vector for M₃ muscarinic acetylcholine receptor gene of bladder smooth muscle cells

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