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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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(a) HR in human cells to convert the chromophore of BFP to the chromophore of EGFP. (b) The sequences of BFP and EGFP near the chromophore-coding regions.
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gks185-F1: (a) HR in human cells to convert the chromophore of BFP to the chromophore of EGFP. (b) The sequences of BFP and EGFP near the chromophore-coding regions.

Mentions: The ARCUT-promoted HR was carried out as shown in Figure 1a. The substrate plasmid pBFP-N1 contains the gene of blue fluorescent protein (BFP), which is under the control of a CMV promoter. On the other hand, the donor DNA codes a part or the whole of enhanced green fluorescent protein (EGFP). Note that these donor DNAs have no initiation codon so that they never express EGFP by themselves. The BFP and the EGFP have the same amino acid sequences except for the amino acids in their chromophore sites (Ser65, His66 and Gly67 for BFP versus Thr65, Tyr66 and Gly67 for EGFP; see Figure 1b). With the use of ARCUT, the chromophore-coding site of the BFP in the substrate DNA was cut, and the scission product was introduced into 293T cells after being purified by gel electrophoresis, together with the donor EGFP fragment. Thus, the amount of the recombinant protein, which is formed in the cells and emits green fluorescence, directly reflects the efficiency of HR reactions in the human cells.Figure 1.


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)

(a) HR in human cells to convert the chromophore of BFP to the chromophore of EGFP. (b) The sequences of BFP and EGFP near the chromophore-coding regions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks185-F1: (a) HR in human cells to convert the chromophore of BFP to the chromophore of EGFP. (b) The sequences of BFP and EGFP near the chromophore-coding regions.
Mentions: The ARCUT-promoted HR was carried out as shown in Figure 1a. The substrate plasmid pBFP-N1 contains the gene of blue fluorescent protein (BFP), which is under the control of a CMV promoter. On the other hand, the donor DNA codes a part or the whole of enhanced green fluorescent protein (EGFP). Note that these donor DNAs have no initiation codon so that they never express EGFP by themselves. The BFP and the EGFP have the same amino acid sequences except for the amino acids in their chromophore sites (Ser65, His66 and Gly67 for BFP versus Thr65, Tyr66 and Gly67 for EGFP; see Figure 1b). With the use of ARCUT, the chromophore-coding site of the BFP in the substrate DNA was cut, and the scission product was introduced into 293T cells after being purified by gel electrophoresis, together with the donor EGFP fragment. Thus, the amount of the recombinant protein, which is formed in the cells and emits green fluorescence, directly reflects the efficiency of HR reactions in the human cells.Figure 1.

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