pMSCV-LTR-dCas9-VP64-BFP

CRISPR Mouse - Activation Neuro-2a Sim1

Experiment
CRISPR Mouse - Activation Neuro-2a Sim1
Product
pMSCV-LTR-dCas9-VP64-BFP from Addgene
Manufacturer
Addgene

Protocol tips

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

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Discussion

Discussion

4 years ago

Author: Mario Udinese Italy

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Discussion

5 years ago

Author: Ben Saar Israel

How to choose a region to target for CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Share your thoughts or question with experts in your field by adding a 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.

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Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

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