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Targeted Transgene Integration
For targeted transgene integration projects, Cellectis bioresearch offers the cGPS® Targeted Integration kits, including all necessary reagents for optimal targeted transgene integration in engineered or native cell lines
Targeted transgene integration is defined as the stable genomic integration of a DNA sequence, most of the time a transgene expression cassette, at a unique genomic position. The unique genomic position is selected to guarantee an adequate transcriptional activity.
Generation of stably expressing cell lines by ‘traditional’ stable transfection protocols usually result in various random integration events in multiple copies (due to plasmid concatemerization) and at various genomic sites. This totally random process leads to high clone genotypic heterogeneity and uncontrolled expression levels. Besides, the level of expression of the randomly integrated transgenes is not stable overtime due to positional effects.
Compared to classical transfection approaches, which can be challenging and time-consuming in order to achieve stable expression, and often require the screening of many clones to overcome clone heterogeneity and low expression levels, cGPS® Targeted Integration kits offer many advantages.
The sequence-specific nuclease binds its recognition site as close as possible to the site of integration, inducing a DSB. The transgene can then be integrated by transfecting a plasmid whose sequence contains both a heterologous region with the transgene and regions homologous to the targeted genomic locus. The DSB is repaired by homologous recombination in presence of this plasmid, referred to as an Integration Matrix (to integrate a modifying DNA sequence).
Any transgene expression cassette can be integrated at the nuclease recognition site.