pcDNA-mC/EBPb

CRISPR Mouse - Activation 3T3-L1 C/EBPβ

Experiment
CRISPR Mouse - Activation 3T3-L1 C/EBPβ
Product
pcDNA-mC/EBPb from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and
the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

Plasmids and Stable Overexpression
3T3-L1 clones, which stably overexpress C/EBPβ, were generated with retrovirus transduction (Platinum Retrovirus Expression System, Pantropic, Cell Biolabs, San Diego, CA). pcDNA-mC/EBPb was a gift from Jed Friedman (Addgene plasmid #49198). The plasmid was digested with and , and subcloned into the retroviral vector, pMXs-IRES-Puro using the - site. The plasmids were transfected to the packaging cell line, and the culture supernatants were collected 24 and 48 hr later, passed through 0.22-μm filters, and added to the cells. Drug selection by 2 μg/ml puromycin was initiated 72 hr after infection. For CRISPR-Cas9 system, pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid #42230). Hif1a target sgRNAs were designed by using CRISPRdirect, and ligated into pX330 plasmid by following the procedures from Zhang lab (http://www.genome-engineering.org/crispr). Sequences are listed in Table . 3T3-L1 cells were co-transfected with the two pX330-Hif1a and a puromycin resistant plasmid which facilitates selection with puromycin (2 μg/ml). Selected colonies went further selection by HREluc activity under hypoxia.

Full paper   Login or join for free to view the full paper.

Reviews

pcDNA-mC/EBPb from Addgene has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

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

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Activation 3T3-L1 C/EBPβ using pcDNA-mC/EBPb from Addgene.

Paper title
Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner
Full paper
Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Addgene for pcDNA-mC/EBPb below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Activation 3T3-L1 C/EBPβ using pcDNA-mC/EBPb from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Become shareholder Discussions About us Contact Privacy Terms