lentiCRISPR

CRISPR Mouse - Deletion RAW 264.7 Tfeb

Experiment
CRISPR Mouse - Deletion RAW 264.7 Tfeb
Product
lentiCRISPR 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

Generation of knockout lines
CRISPR-Cas9 guide RNA targeting sequences for mouse Tfeb and Tfe3 were identified bioinformatically using the CRISPR Design Tool available at https://www.genome-engineering.org.37 The targeting sequence search was limited to the first constitutively expressed exon common to all isoforms of the genes. Targeting sequences used were CACGTACTGTCCACCTCGGC for Tfeb and GAGGCGTGAGCGGCGGGAAC for Tfe3. Targeting sequences were cloned in to the lentiCRISPR plasmid (http://www.addgene.org/49535/) described in Shalem et al.38 Lentivirus was produced for the Tfeb and Tfe3 targeting sequences as well as an empty lentiCRISPR vector for control lines. Lentiviral transfer plasmids were cotransfected with VSV-G envelope (https://www.addgene.org/12259/) and packaging plasmids (https://www.addgene.org/12260/) in to HEK293T cells using Lipofectamine LTX (Invitrogen, 15338–500). Media was changed after 24 h and centrifuged and collected 72 h post-transfection.

RAW 264.7 cells at ∼30% confluency were transduced with hexadimethrine bromide (Sigma Aldrich, 107689) and viruses containing control, Tfeb, Tfe3, or both targeting sequences. Media was removed after 24 h and cells selected with 5 μg/ml puromycin (Sigma Aldrich, P8833). Individual clones were isolated by limiting dilution cloning, and knockouts of TFEB and TFE3 were confirmed via western blotting.

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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 RAW 264.7 Tfeb using lentiCRISPR from Addgene.

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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR below.

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Videos

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