Px330 spcas9

Manufactured by Addgene

PX330-SpCas9 is a plasmid that expresses the Streptococcus pyogenes Cas9 (SpCas9) protein. SpCas9 is a commonly used CRISPR-Cas9 system component for genome editing applications.

Automatically generated - may contain errors

Lab products found in correlation

Aavs1 pur cag egfp, by Addgene (1 mentions) Ab64636, by Abcam (1 mentions) Ab75233, by Abcam (1 mentions) G1544, by Merck Group (1 mentions) Sc 8025, by Santa Cruz Biotechnology (1 mentions) T4026, by Merck Group (1 mentions) Pbabe puro, by Addgene (1 mentions) Ab8227, by Abcam (1 mentions)

2 protocols using px330 spcas9

CRISPR-Mediated Knock-In of eGFP in iPSCs

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The pX330-SpCas9 and AAVS1-Pur-CAG-EGFP plasmid were acquired from Addgene. gRNAs were designed as previously described²⁸ (link) and cloned into BbsI digested Cas9 plasmid. DKC1 A353V mutant and corrected iPSCs were transfected with the gRNA-Cas9 vector and the knock-in vector and plated onto plate coated with Matrigel. After 24 hours, puromycin selection was performed for additional 3 days to select for positively transfected cells. Surviving colonies were picked and expanded. eGFP knock-in was confirmed by PCR analysis using AAVS1-specific primers.²⁸ (link)

Choi Y.J., Kim M.S., Rhoades J.H., Johnson N.M., Berry C.T., Root S., Chen Q., Tian Y., Fernandez RJ I.I.I., Cramer Z., Adams-Tzivelekidis S., Li N., Johnson F.B, & Lengner C.J. (2023). Patient-Induced Pluripotent Stem Cell–Derived Hepatostellate Organoids Establish a Basis for Liver Pathologies in Telomeropathies. Cellular and Molecular Gastroenterology and Hepatology, 16(3), 451-472.

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Wnt Signaling Protein Interactions

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The following plasmids were used: human DVL2–GFP (pEGFP-N1); SNAP–FZD5 (Koo et al., 2012 (link)); LRP6–GFP (Metcalfe et al., 2010 (link)); PX330-spCas9 (Addgene, #43330) for DVL TKO; PX458-spCas9-GFP (Addgene, #48138) for LRP6 KO. DVL2–GFP was subcloned into pBabe PURO (Addgene) to generate stable cell lines. DVL2 mutants were generated by standard procedures and verified by sequencing. The following antibodies were used: anti-tubulin (1:5000; T4026, Sigma), anti-GFP (1:5000; G1544, Sigma), anti-actin (1:5000; ab8227, Abcam), anti-FZD3 (1:2500; ab75233, Abcam), anti-FZD6 (1:5000; ab128916, Abcam), anti-FZD7 (1:1000; ab64636, Abcam), anti-FZD8 (1:3000; ab40012, Abcam), anti-SNAP (1:5000; P9310S, NEB), anti-DVL1 (1:1000; sc8025, Santa Cruz), anti-DVL3 (1:1000; sc26506, Santa Cruz), anti-DVL2 (1:5000; 3216, Cell Signaling), anti-ABC (1:5000; 8814, Cell Signaling), anti-p-LRP6 S1490 (1:2500; 2568, Cell Signaling).

Gammons M.V., Rutherford T.J., Steinhart Z., Angers S, & Bienz M. (2016). Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay. Journal of Cell Science, 129(20), 3892-3902.

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