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A high throughput method for genome-wide analysis of retroviral integration.

Mantovani J, Holic N, Martinez K, Danos O, Perea J - Nucleic Acids Res. (2006)

Bottom Line: 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.VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events.

View Article: PubMed Central - PubMed

Affiliation: Genethon-CNRS-Université d'Evry Val d'Essonne, UMR 8115, Evry, France.

ABSTRACT
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.

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Related in: MedlinePlus

Overview of VITA method. All steps are detailed in ‘Materiel and Methods’. For oligonucleotides information, see Table 1.
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fig1: Overview of VITA method. All steps are detailed in ‘Materiel and Methods’. For oligonucleotides information, see Table 1.

Mentions: Twenty-one nucleotide long tags are enough to unambiguously define a genomic site in 70% of cases. The method described here is designed to extract and analyze such short sequences in the vicinity of retroviral integration sites. It is based in the ability of the MmeI restriction enzyme to cut DNA outside of its recognition site and includes two steps: the production of an ISF library and the extraction of a 21 bp tag from each fragment. Figure 1 gives a general outline of the method and the different oligonucleotides used are listed in Table 1.


A high throughput method for genome-wide analysis of retroviral integration.

Mantovani J, Holic N, Martinez K, Danos O, Perea J - Nucleic Acids Res. (2006)

Overview of VITA method. All steps are detailed in ‘Materiel and Methods’. For oligonucleotides information, see Table 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636494&req=5

fig1: Overview of VITA method. All steps are detailed in ‘Materiel and Methods’. For oligonucleotides information, see Table 1.
Mentions: Twenty-one nucleotide long tags are enough to unambiguously define a genomic site in 70% of cases. The method described here is designed to extract and analyze such short sequences in the vicinity of retroviral integration sites. It is based in the ability of the MmeI restriction enzyme to cut DNA outside of its recognition site and includes two steps: the production of an ISF library and the extraction of a 21 bp tag from each fragment. Figure 1 gives a general outline of the method and the different oligonucleotides used are listed in Table 1.

Bottom Line: 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.VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events.

View Article: PubMed Central - PubMed

Affiliation: Genethon-CNRS-Université d'Evry Val d'Essonne, UMR 8115, Evry, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus