GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit

CRISPR Human - Activation ERBB2

Experiment

CRISPR Human - Activation ERBB2

Product

GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit from Thermo Fisher Scientific

Manufacturer

Thermo Fisher Scientific

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Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

2.4. Establishment of ERBB2+ and ERBB2− Target Cells
The gene encoding ERBB2 was invalidated in HEK293 cells using CRISPR/Cas9 genome editing. Briefly, a guide RNA sequence (TCATCGCTCACAACCAAGTG) was cloned into the nuclease vector GeneArt CRISPR (ThermoFisher Scientific, France) to guide the Cas9 double-stranded DNA endonuclease to a specific site within exon 6 of the ERBB2 gene to isolate ERBB2− HEK293 cells. HEK293− cells were then transfected with an ERBB2 expression vector (InvivoGen, San Diego, CA) using the FuGENE HD transfection reagent (Promega, Madison, WI). Positive clones were enriched using fluorescence-activated cell sorting and a FITC-labelled anti-ERBB2 monoclonal antibody (Abcam, Cambridge, UK). Stable clones were isolated and characterized for ADCC activity in the presence of the iLite effector cells and Herceptin™ (Roche, France) giving rise to the ERBB2+ HEK293 target cell line.

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Discussion

4 years ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Activation ERBB2 using GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit from Thermo Fisher Scientific.

Paper title

A Novel System for the Quantification of the ADCC Activity of Therapeutic Antibodies

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit below.

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Videos

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