pSpCas9(BB)-2A-Puro (PX459) V2.0

CRISPR Mouse - Activation 3T3-L1 Adrb3

Experiment
CRISPR Mouse - Activation 3T3-L1 Adrb3
Product
pSpCas9(BB)-2A-Puro (PX459) V2.0 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

2.2. Plasmids
HA-tagged mouse β3AR coding sequence was amplified by PCR from pGL2-HA-mβ3AR construct, a kind gift from Dr. Sheila Collins [25] and cloned into pcDNA3.1 vector. Ser247-mutated β3AR construct was generated by QuikChange site-directed mutagenesis kit from Agilent Technologies (#200519) based on pcDNA3.1-HA-mβ3AR. CRISPR/Cas9 vector (PX459) was ordered from Addgene (Plasmid #48139). β3AR-targeted Crispr sgRNAs (sense: 5′-CACCGGGCAGAGTCCACCGCTCAAC-3’; antisense: 5′-AAACGTTGAGCGGTGGACTCTGCCC-3′) were synthesized by Integrated DNA Technologies and cloned into PX459 following the protocol provided by Dr. Feng Zhang [26]. Constitutively active ERK2 (ERKca) expression construct was previously described [27].

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Reviews

pSpCas9(BB)-2A-Puro (PX459) V2.0 from Addgene has not yet been reviewed for this experiment

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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 - Activation 3T3-L1 Adrb3 using pSpCas9(BB)-2A-Puro (PX459) V2.0 from Addgene.

Paper title
Phosphorylation of Beta-3 adrenergic receptor at serine 247 by ERK MAP kinase drives lipolysis in obese adipocytes
Full paper
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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-Puro (PX459) V2.0 below.

Download PDF Download manufacturer protocol

Videos

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