pMSCV-LTR-dCas9-VP64-BFP

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

Plasmids
The pMSCV-LTR-dCas9-VP64-BFP vector, encoding a mammalian codon-optimized
Streptococcus pyogenes dCas9 fused to two C-terminal SV40 NLSs and tagBFP along with a
VP64 domain and the U6-sgRNA-CMV-mCherry-T2A-Puro plasmids were used for cell line
transfections (both kind gifts from Drs. Jonathan S. Weissman and Stanley Qi). sgRNAs
(Supplementary Table 1) were cloned using the In-Fusion HD-cloning kit (Clontech) following the
manufacturer’s protocol into the BstXI and XhoI sites. Mouse knockin vectors were generated by
cloning dCas9-VP64 and U6-sgRNA-CMV-mCherry expression cassettes from the
aforementioned vectors into the TARGATT (CAG + Poly A) plasmid (Applied StemCell). For AAV
vectors, pcDNA-dCas9-VP64 (Addgene 47107), and U6-sgRNA-CMV-mCherry-WPREpA were
cloned replacing the Ef1a-FAS-hChR2(H134R)-mCherry-WPRE-pA with that of our U6-sgRNACMV-mCherry-WPREpA into the backbone of pAAV-Ef1a-FAS-hChR2(H134R)-mCherry-WPREpA (Addgene 37090).

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Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Activation Neuro-2a Sim1 using pMSCV-LTR-dCas9-VP64-BFP from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for pMSCV-LTR-dCas9-VP64-BFP below.

Download manufacturer protocol

Videos

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