A high throughput method for genome-wide analysis of retroviral integration.
Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20-25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.
Mantovani
Julie
J
Genethon-CNRS-Université d'Evry Val d'Essonne, UMR 8115, Evry, France.
Holic
Nathalie
N
Martinez
Kelly
K
Danos
Olivier
O
Perea
Javier
J
D045744:Cell Line, Tumor; D002999:Clone Cells; D015723:Gene Library; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D017422:Sequence Analysis, DNA; D016324:Sequence Tagged Sites; D014161:Transduction, Genetic; D016662:Virus Integration