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TALE-Nuclease AAVS1 Targeting

• Pre-validated AAVS1 safe harbor TALE-Nuclease pair
• AAVS1 cloning donor vector with dual GFP and Puro markers
• Positive control donor vector with RFP along with dual GFP/Puro markers
• Routinely integrate transgenes into the same AAVS1 safe harbor locus

Targeted integration at a safe genomic locus
Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences to modulate gene expression. When fused to a FokI cleavage domain, TALE nucleases (TALENs) recognize specific DNA sequences as specified by the internal tandem repeats present in the TALEs. Binding of two TALENs to the target DNA allows FokI to dimerize and create a targeted chromosome break which facilitates homologous recombination at the cleavage site. SBI offers an engineered TALE-Nuclease pair that will target the AAVS1 safe harbor genomic locus, a region of the genome that is considered to be both transcriptionally active and the disruption of which does not lead to discernable phenotypic effects. This TALE-Nuclease pair, when used in conjunction with the AAVS1 donor vectors with homologous recombination flanking arms, enables a powerful and routine method for gene addition to a specific and safe genomic destination.

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AAVS1 recombination validation data
The genomic structure of the targeted AAVS1 locus, with the locations of the exons, TALE-Nuclease cutting site and the left/right homologous recombination arms of the positive control HR donor vector are depicted below. Targeting the AAVS1 locus in human 293T cells with the positive control GE603A-1 vector (lower panel) demonstrates a strict dependence on having the AAVS1 TALE-Nuclease pair added with the control HR donor vector. The targeted 293T cells were treated with puromycin 48 hours after transfection and selected for 5 days. Representative images of GFP and RFP markers show successful insertion.

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AAVS1 locus diagram, sample integration data and junction PCR analysis
Genomic DNA samples (200ng) of human 293T cells were analyzed for AAVS1 targeted homologous recombination using junction PCR on both sides of the HR donor vector (primer sets kit catalog# GE603PR-1). AAVS1 Donor plus AAVS1 TALE-Nuclease pair (Lane 1) and Donor alone without TALE-Nuclease pair (Lane 3) 5´ primer set and the AAVS1 Donor plus AAVS1 TALE-Nuclease pair (Lane 2) and Donor alone without TALE-Nuclease pair (Lane 4) 3´ primer set were tested after 5 days under puromycin selection. The 1.1kb 5´ junction and the 3´ junction 1.5kb PCR bands expected are clearly observed in the AAVS1 donor plus TALE-Nuclease pair and demonstrates accurate homologous recombination at the AAVS1 locus.

 

 

Description Cat# Size Price    
AAVS1 Safe Harbor cDNA/miRNA Donor Vector pAAVS1D-PGK.MCS-EF1a.copGFPpuro GE602A-1-SBI 10 ug 1236 € DETAILS   Add to Cart 
AAVS1 Safe Harbor Positive Control Donor Vector pAAVS1D-CMV.RFP-EF1a.copGFPpuro GE603A-1-SBI 10 ug 1236 € DETAILS   Add to Cart 
5' and 3' AAVS1 Positive Control Donor Vector Primer Mixes for Junction PCR Assays (10 uM) GE603PR-1-SBI 100 ul each 335 € DETAILS   Add to Cart 

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