Jul 11, 2024
Generation of pLKO.1 Plasmids Containing shRNA
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Generation of pLKO.1 Plasmids Containing shRNA. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnm4ygk5/v1
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103190
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Generation of pLKO.1 Plasmids Containing shRNA
**Obtain shRNA Plasmids**
- Obtain pLKO.1 Puro plasmids containing shRNA sequences against mouse/rat Lrrk2 (shLrrk2: TRCN0000322193) and mouse/rat Atg7 (shAtg7: TRCN0000092163) from the RNAi Consortium (TRC) via Dharmacon.
**Synthesize Scrambled shRNA Sequences**
- Generate two scrambled shRNA sequences: GTTGCTGAATGGCGGATCTAT and GTTGCGGTTATGAATAGTACT.
**Cloning into pLKO.1 Vector**
- Clone the scrambled shRNA sequences into the pLKO.1 TRC cloning vector254 following the protocols provided by Addgene (https://www.addgene.org/protocols/plko/).
**pLKO.1-shRNA-EGFP Plasmid Construction**
- Remove CAG-EGFP from pLenLox-shNL1-CAG-EGFP255.
- Insert the CAG-EGFP sequence between the KpnI and SpeI sites in the pLKO.1 Puro vector, replacing the puromycin resistance gene to generate pLKO.1-shRNA-EGFP.
**pLKO.1-shRNA-mCherry Plasmid Construction**
- Replace EGFP with mCherry between the KpnI and NheI sites in the pLKO.1 Puro vector to generate pLKO.1-shRNA-mCherry plasmids.
Notes:
- Validate the integrity and orientation of cloned sequences through restriction digestion and sequencing.
- Use sterile techniques and appropriate safety measures when working with plasmid DNA.