pSpCas9(BB)-2A-GFP (PX458)

CRISPR Mouse - Activation 3T3-L1 ZPR9

Experiment
CRISPR Mouse - Activation 3T3-L1 ZPR9
Product
pSpCas9(BB)-2A-GFP (PX458) 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

Generation of ZPR9 (T252A) knockin (KI) and knockout (KO) cell lines
Genomic mutations in 3T3-L1 or HEK293 cells were generated using the CRISPR/Cas9 system as described previously30,31. In brief, single-guide (sg) RNAs were designed to target the genomic areas adjacent to the ZPR9 mutation site (target sequence, 5′-GCTGAGGAAGGCCCACCCCT-3′). Two complementary oligos (5′-CACCGGCTGAGGAAGGCCCACCCCT-3′ and 5′-AAACAGGGGTGGGCCTTCCTCAGCC-3′) containing the ZPR9 guide sequence and Bbs1 ligation adapters were synthesized by Bioneer Ltd. (Cheongwon, Korea). The annealed oligos were ligated into the Bbs1-digested pX458 vector (Addgene plasmid no. 48138) using the Quick-Ligation system (New England BioLabs, Ipswich, MA, USA). To generate the ZPR9 (T252A) KI cell line, 3T3-L1 cells were cultured in a 24-well plate at ~60% confluence and cotransfected with 1 μg of ZPR9 sg RNA plasmid and pUC19 ZPR9(T252A) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or WelFect-Ex™ Plus (WelGENE, Daegu, Korea). To generate the ZPR9 KO cell line, HEK293 cells were transfected with 1 μg of ZPR9 sg RNA plasmid without pUC19. Forty-eight hours after transfection, the cells were trypsinized and diluted in medium for single-cell seeding in a 96-well plate, and GFP-positive cells were screened, followed by genomic DNA extraction. Exon 2 of ZPR9 was amplified by PCR using a ZPR9-specific PCR primer pair as follows: 5′-ATTGGGAAGATATTGATTCTGATGAAGA-3′ (forward) and 5′-CCAAGTATTTAATCAGT CCCTTAATATCTG-3′ (reverse). The PCR products were A-tailed and cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). The individual ZPR9 (T252A) KI clones were then confirmed by DNA sequencing (Bioneer Ltd., Cheongwon, Korea). On the other hand, the ZPR9 KO clones were validated by immunoblotting with an anti-ZPR9 antibody.

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Reviews

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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 ZPR9 using pSpCas9(BB)-2A-GFP (PX458) from Addgene.

Paper title
Zinc finger protein ZPR9 functions as an activator of AMPK-related serine/threonine kinase MPK38/MELK involved in ASK1/TGF-β/p53 signaling pathways
Full paper
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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-GFP (PX458) below.

Download PDF Download manufacturer protocol

Videos

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