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p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

Herrmann F, Garriga-Canut M, Baumstark R, Fajardo-Sanchez E, Cotterell J, Minoche A, Himmelbauer H, Isalan M - PLoS ONE (2011)

Bottom Line: The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors.We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries.We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation and UPF, Barcelona, Spain.

ABSTRACT
The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

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Episomal gene repair assay in HEK293T cells.(A) Schematic representation of the experimental setup. (B) Cells transfected with repair plasmid, target plasmid and ZFN expression vectors were analyzed after 48 hrs by flow cytometry. The diagram displays the fraction of EGFP-positive cells normalized for transfection efficiency. Statistically significant increases in homologous recombination (HR), compared to non-induced HR control (dashed grey line) are indicated with asterisks (** = p<0.0001 and * = p<0.002).
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pone-0020913-g004: Episomal gene repair assay in HEK293T cells.(A) Schematic representation of the experimental setup. (B) Cells transfected with repair plasmid, target plasmid and ZFN expression vectors were analyzed after 48 hrs by flow cytometry. The diagram displays the fraction of EGFP-positive cells normalized for transfection efficiency. Statistically significant increases in homologous recombination (HR), compared to non-induced HR control (dashed grey line) are indicated with asterisks (** = p<0.0001 and * = p<0.002).

Mentions: To evaluate whether our potential p53-specific ZFNs (z771 and z1166) could promote homologous recombination or gene repair in vivo, they were first tested in a plasmid-based EGFP repair assay, developed by the Cathomen lab [56] (Fig. 4A).


p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

Herrmann F, Garriga-Canut M, Baumstark R, Fajardo-Sanchez E, Cotterell J, Minoche A, Himmelbauer H, Isalan M - PLoS ONE (2011)

Episomal gene repair assay in HEK293T cells.(A) Schematic representation of the experimental setup. (B) Cells transfected with repair plasmid, target plasmid and ZFN expression vectors were analyzed after 48 hrs by flow cytometry. The diagram displays the fraction of EGFP-positive cells normalized for transfection efficiency. Statistically significant increases in homologous recombination (HR), compared to non-induced HR control (dashed grey line) are indicated with asterisks (** = p<0.0001 and * = p<0.002).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111460&req=5

pone-0020913-g004: Episomal gene repair assay in HEK293T cells.(A) Schematic representation of the experimental setup. (B) Cells transfected with repair plasmid, target plasmid and ZFN expression vectors were analyzed after 48 hrs by flow cytometry. The diagram displays the fraction of EGFP-positive cells normalized for transfection efficiency. Statistically significant increases in homologous recombination (HR), compared to non-induced HR control (dashed grey line) are indicated with asterisks (** = p<0.0001 and * = p<0.002).
Mentions: To evaluate whether our potential p53-specific ZFNs (z771 and z1166) could promote homologous recombination or gene repair in vivo, they were first tested in a plasmid-based EGFP repair assay, developed by the Cathomen lab [56] (Fig. 4A).

Bottom Line: The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors.We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries.We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation and UPF, Barcelona, Spain.

ABSTRACT
The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

Show MeSH
Related in: MedlinePlus