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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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Correlation between ISF clones/tags and genes per chromosome. ISFs (607 ISFs) and one-hit tags (1349 tags) obtained by VITA technique on HeLa cells were mapped into human genome. Dark grey boxes and clear grey boxes represent the percentage of ISF and tags identified per chromosome respectively. White boxes represent the percentage of genes per chromosome.
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fig4: Correlation between ISF clones/tags and genes per chromosome. ISFs (607 ISFs) and one-hit tags (1349 tags) obtained by VITA technique on HeLa cells were mapped into human genome. Dark grey boxes and clear grey boxes represent the percentage of ISF and tags identified per chromosome respectively. White boxes represent the percentage of genes per chromosome.

Mentions: Lentiviral vectors are known to integrate preferentially into transcribed regions of the genome (5). As a further validation of our method, we checked whether a similar bias was observed in the ISF library and in the tag populations. Additional HeLa ISF library clones (n = 932) were sequenced and 607 insertion sites could be identified. As expected, the number of insertion sites found on each chromosome was correlated with the number of genes (Pearson's correlation rate = 0.79, P-value = 2.072e−6). The same correlation was observed with the 1349 ‘one-hit’ tags described in HeLa tag library (Pearson's correlation rate = 0.86, P-value = 2.84e−8). These results suggest that the tag population properly reflects the actual integrome (Figure 4).


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)

Correlation between ISF clones/tags and genes per chromosome. ISFs (607 ISFs) and one-hit tags (1349 tags) obtained by VITA technique on HeLa cells were mapped into human genome. Dark grey boxes and clear grey boxes represent the percentage of ISF and tags identified per chromosome respectively. White boxes represent the percentage of genes per chromosome.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Correlation between ISF clones/tags and genes per chromosome. ISFs (607 ISFs) and one-hit tags (1349 tags) obtained by VITA technique on HeLa cells were mapped into human genome. Dark grey boxes and clear grey boxes represent the percentage of ISF and tags identified per chromosome respectively. White boxes represent the percentage of genes per chromosome.
Mentions: Lentiviral vectors are known to integrate preferentially into transcribed regions of the genome (5). As a further validation of our method, we checked whether a similar bias was observed in the ISF library and in the tag populations. Additional HeLa ISF library clones (n = 932) were sequenced and 607 insertion sites could be identified. As expected, the number of insertion sites found on each chromosome was correlated with the number of genes (Pearson's correlation rate = 0.79, P-value = 2.072e−6). The same correlation was observed with the 1349 ‘one-hit’ tags described in HeLa tag library (Pearson's correlation rate = 0.86, P-value = 2.84e−8). These results suggest that the tag population properly reflects the actual integrome (Figure 4).

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