shRNA 以及逆转录载体pSUPER和脂质体介导的稳定干扰

Silvia Soddu

doi:10.21769/BioProtoc.243

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Peer-reviewed

Stable Interference by shRNA with pSUPER.retro Vectors and Lipofectamine

shRNA 以及逆转录载体pSUPER和脂质体介导的稳定干扰

SS Silvia Soddu email

发布: 2012年08月05日第2卷第15期 DOI: 10.21769/BioProtoc.243 浏览次数: 14080

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实验方案合集

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Abstract

RNA interference is a powerful genetic approach for efficiently silencing target genes. Expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes. This protocol describes a method to stably integrate shRNA constructs with pSUPER.retro vectors and Lipofectamine in RKO and H1299 cells. This method can be applied to cells from other lines with modification of drug selection and cell conditions. pSUPER.retro vectors can be also transfected into packaging cells by this same method to produce retroviral supernatants.

Keywords: Interference (干扰)

ShRNA (shRNA)

Transfection (转染)

Materials and Reagents

Lipofectamine/plus transfection reagents (Life Technologies, Invitrogen™, catalog number: 11514-015 )
Dulbecco’s modification of eagles medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 21885025 )
Trypsin-EDTA (Life Technologies, Invitrogen™, catalog number: 25300-054 )
FBS (Life Technologies, Invitrogen™, catalog number: 10099-141 )
Puromycin (Sigma-Aldrich, catalog number: P8833 )
DPBS (Lonza, catalog number: BE17-512F )
pSUPER.retro.puro vector (OligoEngine, catalog number: VEC-PRT-0002 )