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Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter.

Katada H, Harumoto T, Shigi N, Komiyama M - Nucleic Acids Res. (2012)

Bottom Line: A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp).Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold.It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.

ABSTRACT
A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

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Effects of the homology length on the ARCUT-mediated HR efficiency in human cells. (a) The substrate DNA coding BFP and the donor DNAs having different lengths of homology regions. (b) Typical fluorescence microscopy images of the 293T cells cultured for 48 h after the transfection with the ARCUT-treated substrate DNA and the donor EGFP long (717 bp). The upper and lower panels show blue channel (Ex: 360 nm, Em: 470 nm) and green channel (Ex: 480 nm, Em: 520 nm), respectively. (c) The fraction of green fluorescence-emitting cells in the total cells measured by flow cytometry. The terms L and S refer to the donor EGFP long and the donor EGFP short, respectively.
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gks185-F3: Effects of the homology length on the ARCUT-mediated HR efficiency in human cells. (a) The substrate DNA coding BFP and the donor DNAs having different lengths of homology regions. (b) Typical fluorescence microscopy images of the 293T cells cultured for 48 h after the transfection with the ARCUT-treated substrate DNA and the donor EGFP long (717 bp). The upper and lower panels show blue channel (Ex: 360 nm, Em: 470 nm) and green channel (Ex: 480 nm, Em: 520 nm), respectively. (c) The fraction of green fluorescence-emitting cells in the total cells measured by flow cytometry. The terms L and S refer to the donor EGFP long and the donor EGFP short, respectively.

Mentions: In Figure 3a, two donor DNAs of different lengths were used. The donor EGFP long (717 bp) contains a homology sequence of 185 bp in the upstream of the chromophore-coding site and a homology sequence of 513 bp in the downstream. On the other hand, the donor EGFP short (119 bp) contains homology sequences of 50 bp in both the upstream and the downstream. With the use of ARCUT-3′-overhangs (also with ARCUT-5′-overhangs), HR was efficient as evidently shown by the emission of green fluorescence from the recombinant protein (Figure 3b). Without the ARCUT scission, however, HR hardly took place. The number of green fluorescence-emitting cells was determined by flow cytometric analysis (Figure 3c). When the donor EGFP long (L) was used, the fraction of green fluorescence-emitting cells in the total cells was 22.5%. With the use of the donor EGFP short (S), the fraction was slightly lower (11.1%). As was previously reported, a longer donor is more favorable for HR (26). However, it is noteworthy that, without the ARCUT treatment, HR is much less efficient with EGFP long and virtually nil with EGFP short. Essential role of ARCUT for efficient HR is reconfirmed.Figure 3.


Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter.

Katada H, Harumoto T, Shigi N, Komiyama M - Nucleic Acids Res. (2012)

Effects of the homology length on the ARCUT-mediated HR efficiency in human cells. (a) The substrate DNA coding BFP and the donor DNAs having different lengths of homology regions. (b) Typical fluorescence microscopy images of the 293T cells cultured for 48 h after the transfection with the ARCUT-treated substrate DNA and the donor EGFP long (717 bp). The upper and lower panels show blue channel (Ex: 360 nm, Em: 470 nm) and green channel (Ex: 480 nm, Em: 520 nm), respectively. (c) The fraction of green fluorescence-emitting cells in the total cells measured by flow cytometry. The terms L and S refer to the donor EGFP long and the donor EGFP short, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367209&req=5

gks185-F3: Effects of the homology length on the ARCUT-mediated HR efficiency in human cells. (a) The substrate DNA coding BFP and the donor DNAs having different lengths of homology regions. (b) Typical fluorescence microscopy images of the 293T cells cultured for 48 h after the transfection with the ARCUT-treated substrate DNA and the donor EGFP long (717 bp). The upper and lower panels show blue channel (Ex: 360 nm, Em: 470 nm) and green channel (Ex: 480 nm, Em: 520 nm), respectively. (c) The fraction of green fluorescence-emitting cells in the total cells measured by flow cytometry. The terms L and S refer to the donor EGFP long and the donor EGFP short, respectively.
Mentions: In Figure 3a, two donor DNAs of different lengths were used. The donor EGFP long (717 bp) contains a homology sequence of 185 bp in the upstream of the chromophore-coding site and a homology sequence of 513 bp in the downstream. On the other hand, the donor EGFP short (119 bp) contains homology sequences of 50 bp in both the upstream and the downstream. With the use of ARCUT-3′-overhangs (also with ARCUT-5′-overhangs), HR was efficient as evidently shown by the emission of green fluorescence from the recombinant protein (Figure 3b). Without the ARCUT scission, however, HR hardly took place. The number of green fluorescence-emitting cells was determined by flow cytometric analysis (Figure 3c). When the donor EGFP long (L) was used, the fraction of green fluorescence-emitting cells in the total cells was 22.5%. With the use of the donor EGFP short (S), the fraction was slightly lower (11.1%). As was previously reported, a longer donor is more favorable for HR (26). However, it is noteworthy that, without the ARCUT treatment, HR is much less efficient with EGFP long and virtually nil with EGFP short. Essential role of ARCUT for efficient HR is reconfirmed.Figure 3.

Bottom Line: A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp).Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold.It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.

ABSTRACT
A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

Show MeSH