pX333-sgNF1-sgTrp53-Cas9

CRISPR Mouse - Deletion 3T3-L1 Trp53

Experiment
CRISPR Mouse - Deletion 3T3-L1 Trp53
Product
pX333-sgNF1-sgTrp53-Cas9 from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Plasmids and adenoviral vectors
The Ad-sgTrp53-Cre adenoviral vector was constructed as follows. The pX333 vector was kindly provided by A. Ventura (Memorial Sloan Kettering Cancer Center). The Cas9 gene in pX333 was replaced by Cre recombinase (Addgene 60229) using AgeI and EcoRI sites by standard cloning methods. For cloning pX333-sgTrp53-Cre, the pX333-Cre vector was digested with BsaI enzyme and ligated to annealed sgRNA oligonucleotides (Supplementary Table 5) targeting mouse Trp53 exon 7. In the final step, pX333-sgTrp53-Cre vector was cloned into the Ad5 adenoviral shuttle vector, which was kindly provided by A. Ventura (Memorial Sloan Kettering Cancer Center) using XhoI and EcoRI sites. The pcDNA3-EGFP vector was purchased from Addgene (13031). Restriction enzymes and T4 ligase were purchased from New England Biolabs. The pX333-sgNF1-sgTrp53-Cas9 adenoviral vector was constructed using a similar approach. Recombinant adenoviruses were generated by Viraquest Inc.



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

Reviews

pX333-sgNF1-sgTrp53-Cas9 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

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 Trp53 using pX333-sgNF1-sgTrp53-Cas9 from Addgene.

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

Manufacturer protocol

Download the product protocol from Addgene for pX333-sgNF1-sgTrp53-Cas9 below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Deletion 3T3-L1 Trp53 using pX333-sgNF1-sgTrp53-Cas9 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.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms