pX330-U6-Chimeric_BB-CBh-hSpCas9

CRISPR Mouse - Activation 3T3-L1 eIF5A1

Experiment
CRISPR Mouse - Activation 3T3-L1 eIF5A1
Product
pX330-U6-Chimeric_BB-CBh-hSpCas9 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

Plasmid constructs
ERT2CreERT2 Was PCR amplified using pCAG-ERT2CreERT2 (Addgene # 13777) as a template and
was cloned between XhoI and NotI sites of the bicistronic pEFIRES vector (31) harboring a
selectable marker conferring resistance to G418.
To generate pX330-5A1, a 19-nt guide sequence (Table 1) targeting exon 1 of the mouse eIF5A1
gene was ligated into a pX330 hSpCas9 plasmid (Addgene # 42230).
pE-eIF5A1smut, containing a FLAG-tagged eIF5A1 that is refractory to the guide RNA, was
constructed in two steps; first, eIF5A1 cDNA was amplified by PCR using a 5’ primer containing a
LoxP site and appending a FLAG tag to the N-terminus of eIF5A1 and a 3’ primer containing a LoxP
site. The resulting fragment was cloned into the bicistronic pEFIRES vector containing a selectable
marker conferring puromycin resistance (Table 2). Next, three silent mutations were introduced at the
site of recognition by the guide RNA to make it refractory to editing by the CRISPR-Cas9 (Table 2).

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Reviews

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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

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Activation 3T3-L1 eIF5A1 using pX330-U6-Chimeric_BB-CBh-hSpCas9 from Addgene.

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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

Download PDF Download manufacturer protocol

Videos

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