lentiCRISPR v2

CRISPR Mouse - Deletion 3T3-L1 TEAD

Experiment
CRISPR Mouse - Deletion 3T3-L1 TEAD
Product
lentiCRISPR v2 from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
There is no need to perform a negative control golden-gate reaction (without
insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Generation of Knockout Cells Using CRISPR/Cas9 Genome Editing
pSpCas9(BB)-2A–Puro (PX459; Addgene plasmid #48139) and lentiCRISPRv2 (Addgene plasmid #52961) were gifts from Dr. Feng Zhang (Ran et al., 2013; Sanjana et al., 2014). The 20 nucleotide guide sequences were designed using the CRISPR design tool at http://www.genome-engineering.org/crispr or were taken from archived guide sequences from the Genome-scale CRISPR knock-out (GeCKO2) library (Sanjana et al., 2014). Single guide RNAs (sgRNAs) were cloned into PX459 expression vector containing the human codon-optimized Cas9. HEK293A cells were transfected using PolyJet DNA in vitro Transfection Reagent according to the manufacturer’s instructions. In addition, sgRNAs were cloned into the lentiviral vector lentiCRISPRv2. Lentivirus was prepared in 293T cells, and the viral supernatant was collected, and added to MEFs or 3T3-L1 cells with polybrene. 16 hr after infection, the media was changed. 24 hr post transfection, cells were selected with puromycin for 2–3 days. Following removal of puromycin, cells were allowed to recover in regular growth media for 24 hr before being single-cell sorted by FACs (UCSD; Human Embryonic Stem Cell Core, BDInflux) into 96-well plate format. Single clones were expanded and screened by protein immunoblotting, genomic sequencing, and functional assays.

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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 - Deletion 3T3-L1 TEAD using lentiCRISPR v2 from Addgene.

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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

Download PDF Download manufacturer protocol

Videos

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