GeneArt™ Genomic Cleavage Selection Kit

Description

The GeneArt® Genomic Cleavage Selection Kit is a rapid and reliable tool for detecting functionality of engineered nucleases in transfected cells, as well as enriching for modified cells. When using engineered nucleases to create double-stranded breaks in genomic DNA, it is useful to know whether or not the designed nucleases are functional. A way to enrich for the edited cells is also needed, especially when low cleavage activity is suspected or difficult to transfect cell lines are used. The GeneArt® Genomic Cleavage Selection Kit contains a vector with the orange fluorescent protein (OFP) gene for a quick visual check on the functionality of the engineered nuclease. In addition, the reporter genes OFP and CD4 can be used to enrich for edited cells. The vector is co-transfected with genome editing tools such as zinc-finger nuclease (ZFN), transcriptional activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) with results 24-72 hours post transfection.

• Screen for functionality of your engineered nucleases as early as 24 hours post-transfection using fluorescence microscopy
• Enrich for modified cells using fluorescence activated cell sorting (FACS) or Dynabeads® CD4 magnetic beads

OFP reporter and CD4 enrichment
The GeneArt® Genomic Cleavage Selection vector contains an orange fluorescent protein (OFP) reporter gene for quick screening of the functionality of the engineered nucleases in the transfected cells. In addition, the OFP and CD4 reporters can be used for enrichment of the nuclease-modified cells using fluorescence activated cell sorting (FACS) or CD4 antibody conjugated Dynabeads®. The vector has been constructed such that the N-terminal and C-terminal ends of the OFP gene are separated by a cloning site for the target sequence of the programmable nuclease. The upstream sequence coding for the N-terminal portion of the OFP gene contains a region complementary to the 5’ end of the C-terminal region of the OFP gene. Stop codons after the N-terminal OFP sequence ensure no expression of the reporter prior to nuclease activity. The CD4 gene is out of frame for expression when the OFP gene is interrupted by the cloning site.

Workflow
Prior to transfection, two single-stranded DNA oligonucleotides complementary to each other and to the genomic sequence to be edited must be annealed and cloned into the GeneArt® Genomic Cleavage Selection Kit vector. The vector plus genome editing technology is then transfected into the cell. When a double-strand break is introduced into the target sequences in the genome and selection vector by the programmable nuclease, the complementary strands from each end sequence of the vector OFP gene will recombine to restore OFP expression and the CD4 gene is now in frame for expression. Thus, cleavage by TALEN, CRISPR, or ZFN can be checked as early as 24 h post transfection by simply viewing the transfected cells under a fluorescence microscope. The percentage of OFP positive cells will reflect the cleavage activity of TALEN, CRISPR, or ZFN. The OFP positive cells can be enriched through FACS. Additionally, the membrane protein CD4 coding gene is fused to OFP through T2A self-cleavage peptide, so the nuclease-modified cells can be enriched using Dynabeads® with CD4 antibody. Thus, the GeneArt® Genomic Cleavage Selection Kit allows for simple, rapid evaluation of the functionality of the programmable nuclease and direct enrichment of the genome-modified cells.

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Spécifications

Cloning Method:	Restriction Enzyme ⁄ MCS
Product Line:	GeneArt®
Product Size:	10 reactions
Shipping Condition:	Dry Ice

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