pCas-Guide-EF1a-GFP

CRISPR Mouse - Deletion αT3 Stim2

Experiment
CRISPR Mouse - Deletion αT3 Stim2
Product
pCas-Guide-EF1a-GFP 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

4.3. Generation and Analysis of STIM2 Knock-Out (KO) Cell Lines
The CRISPR-Cas9 system was used to target exons in the mouse Stim2 gene. Oligonucleotides targeting exon 2 (5′-CAAGGACGGCGGGATCGAAG-3′) were annealed and ligated into AflII-linearized gRNA vector (Addgene, Cambridge, MA, USA). Alternatively, exon 8 (5′-GATGCAGCTAGCCATCGCTA-3′) was targeted to confirm our findings from our exon 2 KO. CRISPR target sequences were searched using NCBI BLAST to verify specificity. Following molecular cloning to insert our target sequences, the CRISPR gRNA vectors were sequenced for verification. Lipofectamine 2000 was used to transfect cells with a mixture of the Stim2-targeted gRNA construct, and vectors encoding hCas9 (Addgene, Cambridge, MA, USA) and EGFP-c1. In the αT3 cells, the same exon 2 target sequence was used, but cloned into a polycistronic vector (pCas-Guide-EF1a-GFP) that also contained hCas9 and GFP (OriGene, Rockville, MD, USA, Cat. No. GE100018). In the polycistronic vector we also cloned in a 20 base-pair scramble sequence to generate controls (5′-GTCGCTTGGGCGAGAGTAAG-3′). Two days post-transfection, EGFP-expressing cells were selected by fluorescence-activated cell sorting and plated one cell per well in a 96-well plate. Colonies were expanded and screened for knock-out of STIM2 expression by Western immunoblotting with anti-STIM2 (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 4917) as described above.

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Discussion

Discussion

1 year 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

2 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

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Manufacturer protocol

Download the product protocol from Addgene for pCas-Guide-EF1a-GFP below.

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Videos

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