Off-targeted Sites Analysis Of Positive Cells Targeted By Zinc-finger Nucleases

Zinc-finger nucleases induce a targeted DNA double-strand break(DSB) at a specificgenomic locus and gene editing in living cells.However,methods which can prove thegenome-wide specificity of ZFN is still limited. We show that nonhomologous end-joiningcaptures integrase-defective lentiviral vectors(IDLV) at DSBs,tagging these transientevents.Genome-wide integration site analysis mapped the actual in vivo cleavage activity ofZFN pair targeting CSN2.DSBs are repaired by nonhomologous end-joining(NHEJ),which often introducesinserted or deleted sequences(indels) at the sealed break,or by homology-directedrepair(HDR),which faithfuLly restores the original sequence by copying it from the sisterchromatid.Here we have addressed this challenge by using integrase-defecive lentiviralvectors(IDLV).We hypothesized that linear double-stranded IDLV genomes present in thenucleus of trandsduced cells couLd be ligated into DSBs by NHEJ，thereby stably taggingtransient,otherwise undetectable DSBs.So you just need to monitor integration sites of IDLVto reflect transgene integration of ZFN.This experiment’s mainly experimental contents and resuLts are as follosws:1,We built a integrase-defective lentiviral vector with neo gene.2,Using a Lenti-X HTX Packaging System to generate replication-incompetentvirions.Using lentiviral supernatants and ZFN transfect bovine fetal fibroblasts at the sametime，another comparison only IDLV incubation, and several stably integrated clones wereobtained by G418screening. Analyzed fluorescence of monoclonal by flow cytometry(FCM)to show that the efficiency, transfected at the same time with lentiviral supermatants andZFN,is much higher than IDLV alone.This resuLt can demonstrate that DSBs mediated byZFN has been caught by IDLV.3,Extracted genome from monoclonal cells for off-targeted analysis.We detected60off-targeted sites by using LAMPCR,which has8clustered integration sites and definedoff-targeted hot sites.4,Off-targeted analysis for targeted positive monoclonal.We extract genome from16 targeted positive monoclonal,design primers respectively countered with8off-targeted hotsites,amplificate gene sequences of these sites by PCR. The resuLt show that,only onemonoclonal was random integration insert,other monoclonal has no random integration orpoint mutation.ZFN is suitable for nuclear transfer to product transgenic animals.