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Stimulation of oligonucleotide-directed gene correction by Redβ expression and MSH2 depletion in human HT1080 cells.

Xu K, Stewart AF, Porter AC - Mol. Cells (2014)

Bottom Line: Here we show that Redβ expression is well tolerated in a human cell line where it consistently promotes ssOR.Furthermore, we find that the effects of Redβ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity.These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China ; Gene Targeting Group, Department of Hematology, Faculty of Medicine, Imperial College London, London W12 0NN, UK.

ABSTRACT
The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of Redβ, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that Redβ expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of Redβ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

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Reagents used for Redβ expres sion and ssOR assays. (A) Structure of Redβ expression plasmid. (B) Structure of plasmid carrying the neo* target for gene correction. (C) Sequence surrounding the 4 bp insertion (bold underlined) in neo*. (D) Sequence of control (CO) and repair (RO) ssOs used in ssOR assays. (E) Immunoblot analysis of Redβ expression in hygroR clones isolated after cotransfection of HT1080 cells with the plasmids shown in (A) and (B). Clones were expanded and grown continually with tetracycline (Tet) in the growth medium (+), or for 3 days after the removal of tetracycline (−), before analysis.
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f1-molcell-38-1-33: Reagents used for Redβ expres sion and ssOR assays. (A) Structure of Redβ expression plasmid. (B) Structure of plasmid carrying the neo* target for gene correction. (C) Sequence surrounding the 4 bp insertion (bold underlined) in neo*. (D) Sequence of control (CO) and repair (RO) ssOs used in ssOR assays. (E) Immunoblot analysis of Redβ expression in hygroR clones isolated after cotransfection of HT1080 cells with the plasmids shown in (A) and (B). Clones were expanded and grown continually with tetracycline (Tet) in the growth medium (+), or for 3 days after the removal of tetracycline (−), before analysis.

Mentions: Because Redβ expression could be deleterious in mammalian cells, we expressed Redβ stably but inducibly. The Redβ open-reading-frame was cloned, with an N-terminal nuclear localisation signal (nls), downstream of a tetracycline responsive element (TRE) to generate pTRE-Redβ (Fig. 1A). To provide a target gene for ssOR, as well as a drug resistance marker for the selection of stable transfectants, a second plasmid, pcDNA/PGKneo* was used (Fig. 1B). It carries two expression cassettes, one conferring resistance to hygromycin, the other (PGKneo*) capable of conferring resistance to G418 only after correction of a 4 bp insertion in its neomycin phosphotransferase (neo) coding sequence. The DNA sequence of this region, and of repair and control ssOs used in ssOR assays, are shown in Figs. 1C and 1D. Plasmids pTRE-Redβ and pcDNA/PGKneo* were co-transfected, the former in molar excess, into an HT1080 derivative [Rht14; (Brough et al., 2011)] expressing an improved tetracycline transactivator protein capable of driving transcription from the TRE. Hygromycin-resistant colonies were expanded and analysed for Redβ expression by immunoblotting. Several clones showing tetracycline-regulated Redβ expression were identified and immunoblots for some are shown in Fig. 1E.


Stimulation of oligonucleotide-directed gene correction by Redβ expression and MSH2 depletion in human HT1080 cells.

Xu K, Stewart AF, Porter AC - Mol. Cells (2014)

Reagents used for Redβ expres sion and ssOR assays. (A) Structure of Redβ expression plasmid. (B) Structure of plasmid carrying the neo* target for gene correction. (C) Sequence surrounding the 4 bp insertion (bold underlined) in neo*. (D) Sequence of control (CO) and repair (RO) ssOs used in ssOR assays. (E) Immunoblot analysis of Redβ expression in hygroR clones isolated after cotransfection of HT1080 cells with the plasmids shown in (A) and (B). Clones were expanded and grown continually with tetracycline (Tet) in the growth medium (+), or for 3 days after the removal of tetracycline (−), before analysis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4314130&req=5

f1-molcell-38-1-33: Reagents used for Redβ expres sion and ssOR assays. (A) Structure of Redβ expression plasmid. (B) Structure of plasmid carrying the neo* target for gene correction. (C) Sequence surrounding the 4 bp insertion (bold underlined) in neo*. (D) Sequence of control (CO) and repair (RO) ssOs used in ssOR assays. (E) Immunoblot analysis of Redβ expression in hygroR clones isolated after cotransfection of HT1080 cells with the plasmids shown in (A) and (B). Clones were expanded and grown continually with tetracycline (Tet) in the growth medium (+), or for 3 days after the removal of tetracycline (−), before analysis.
Mentions: Because Redβ expression could be deleterious in mammalian cells, we expressed Redβ stably but inducibly. The Redβ open-reading-frame was cloned, with an N-terminal nuclear localisation signal (nls), downstream of a tetracycline responsive element (TRE) to generate pTRE-Redβ (Fig. 1A). To provide a target gene for ssOR, as well as a drug resistance marker for the selection of stable transfectants, a second plasmid, pcDNA/PGKneo* was used (Fig. 1B). It carries two expression cassettes, one conferring resistance to hygromycin, the other (PGKneo*) capable of conferring resistance to G418 only after correction of a 4 bp insertion in its neomycin phosphotransferase (neo) coding sequence. The DNA sequence of this region, and of repair and control ssOs used in ssOR assays, are shown in Figs. 1C and 1D. Plasmids pTRE-Redβ and pcDNA/PGKneo* were co-transfected, the former in molar excess, into an HT1080 derivative [Rht14; (Brough et al., 2011)] expressing an improved tetracycline transactivator protein capable of driving transcription from the TRE. Hygromycin-resistant colonies were expanded and analysed for Redβ expression by immunoblotting. Several clones showing tetracycline-regulated Redβ expression were identified and immunoblots for some are shown in Fig. 1E.

Bottom Line: Here we show that Redβ expression is well tolerated in a human cell line where it consistently promotes ssOR.Furthermore, we find that the effects of Redβ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity.These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China ; Gene Targeting Group, Department of Hematology, Faculty of Medicine, Imperial College London, London W12 0NN, UK.

ABSTRACT
The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of Redβ, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that Redβ expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of Redβ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

Show MeSH
Related in: MedlinePlus