Jul 10, 2024

Public workspaceshRNA plasmids

DOI
dx.doi.org/10.17504/protocols.io.yxmvm288bg3p/v1
  • 1Duke University
Shiyi Wang
Open access
DOI: dx.doi.org/10.17504/protocols.io.yxmvm288bg3p/v1
Document Citation: Shiyi Wang 2024. shRNA plasmids. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm288bg3p/v1
License: This is an open access document distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: May 23, 2023
Last Modified: July 10, 2024
Document Integer ID: 82336
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Abstract
How to make shRNA plasmids
1. pLKO.1 Puro plasmids containing shRNA (pLKO.1-shRNA) against mouse/ratLrrk2(shLrrk2: TRCN0000322193; GGCCGAGTTGTGGATCATATT), was obtained from the RNAi Consortium (TRC) via Dharmacon.

2. A scrambled shRNA sequence was generated (GTTGCTGAATGGCGGATCTAT) and cloned into the pLKO.1 TRC cloning vector according to Addgene protocols (https://www.addgene.org/protocols/plko/).

3. To generate pLKO.1 shRNA plasmids that express EGFP (pLKO.1-shRNA-EGFP), CAG-EGFP was removed from pLenLox-shNL1-CAG-EGFP and inserted between Kpn1 and SpeI sites in pLKO.1 Puro, replacing the puromycin resistance gene. pLKO.1 shRNA mCherry plasmids were generated by replacing EGFP with mCherry between KpnI and NheI sites.