A: No, the only sure way to determine the true percentage and nature of genomic modifications is by sequencing verification
A1: GeneArt Genomic Cleavage Detection Kit. Yes, the genomic cleavage detection (GCD) assay provides a reliable estimate of genomic modification efficiency, affording statistical guidance as to the requisite number of clones to isolate and analyze in order to obtain the desired mutation. Additionally, the GCD assay is compatible with Sanger and Ion Personal Genome Machine (PGM) sequencing.
A2: GeneArt Genomic Cleavage Selection Kit. Yes, the surrogate reporter contains an enrichment functionality allowing for the enrichment of cells with genetic modifications. Although CD4 and orange fluorescent protein (OFP) expression does not guarantee corresponding genomic modification, it greatly increases the probability of recovering nuclease-modified cells.
A: We have observed up to 30-fold enrichment of genetically modified cells as confirmed by the GCD assay. This effectively reduces the number of clones needed to screen.
A: Yes, there is the potential for background as there is the possibility for endogenous mutations and spurious digestion products. However the primers are designed to give specific and unique cleavage products that allow for the accurate detection of the desired product.
A: As with any fluorescent or staining-based cell assay, auto-fluorescence and background staining are possible and the proper controls must be used to account and subtract for this potential background. That being said, the vector was designed and the product requirements set such that the positive fluorescent readout is clearly differentiable from any background.
A: The OFP and CD4 functionalities were added to the vector to provide customers flexibility in using their desired enrichment method. As a rule, bead enrichment is gentler and less expensive than cell-sorting, however sorting typically offers more stringent enrichment. Additionally, individual cell lines vary greatly in their expression of fluorescent and membrane proteins. The two functionalities provide versatility.