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I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

Fenina M, Simon-Chazottes D, Vandormael-Pournin S, Soueid J, Langa F, Cohen-Tannoudji M, Bernard BA, Panthier JJ - PLoS ONE (2012)

Bottom Line: The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells.However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus.These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Mouse functional Genetics, Institut Pasteur, Paris, France.

ABSTRACT
Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

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H2B-mCherry gene targeting by HR at the Dct locus.(A) Insertion of H2B-mCherry gene at the Dct locus. The DctI-SceI allele, the HR2 repair vector and the DctH2B-mCherry-Neo allele are represented from top to bottom. AvrII sites are indicated. There are no homologous sequences between DctI-SceI and HR2 close to the I-SceI site. A lightning denotes I-SceI expression from pCMV-I-SceI or pCAG-I-SceI plasmid. The 1.4 and 4.5 kb of Dct isogenic DNA are depicted by grey rectangles. (B) Southern blot analysis of Dct+/+, Dct I-SceI/+ES cells, and DctH2B-mCherry-Neo/+ ES clones 7E and 11E. ES clones 7E and 11E were obtained after transfection with the linear HR2 repair vector, and with both the supercoiled HR2 repair vector and pCAG-I-SceI expression plasmid, respectively. Genomic DNAs were digested with AvrII. The probe used for the hybridization is the external 5′ probe depicted by a black bar. The 3.9 kb fragment is carried by the Dct + and Dct I-SceI alleles, and the 6.5 kb fragment is distinctive of the Dct H2B-mCherry-Neo targeted allele.
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pone-0039895-g005: H2B-mCherry gene targeting by HR at the Dct locus.(A) Insertion of H2B-mCherry gene at the Dct locus. The DctI-SceI allele, the HR2 repair vector and the DctH2B-mCherry-Neo allele are represented from top to bottom. AvrII sites are indicated. There are no homologous sequences between DctI-SceI and HR2 close to the I-SceI site. A lightning denotes I-SceI expression from pCMV-I-SceI or pCAG-I-SceI plasmid. The 1.4 and 4.5 kb of Dct isogenic DNA are depicted by grey rectangles. (B) Southern blot analysis of Dct+/+, Dct I-SceI/+ES cells, and DctH2B-mCherry-Neo/+ ES clones 7E and 11E. ES clones 7E and 11E were obtained after transfection with the linear HR2 repair vector, and with both the supercoiled HR2 repair vector and pCAG-I-SceI expression plasmid, respectively. Genomic DNAs were digested with AvrII. The probe used for the hybridization is the external 5′ probe depicted by a black bar. The 3.9 kb fragment is carried by the Dct + and Dct I-SceI alleles, and the 6.5 kb fragment is distinctive of the Dct H2B-mCherry-Neo targeted allele.

Mentions: We thus decided to remove the two short regions of homology (including the loxP site) and to construct a novel repair vector. Therefore, a second destination vector (DV2) was produced using the H2B-mCherry reporter gene [31]. The HR2 repair vector was constructed (Fig. 2). It carries H2B-mCherry, a NeoR cassette flanked with FRT sites, two regions of homology with the DctI-SceI allele, 1.4 and 4.5 kb in length, and a HSV-TK negative selection cassette (Fig. 5A). By contrast with HR1, HR2 displays neither a short region of homology with the Dct gene next to the I-SceI site nor a loxP site.


I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

Fenina M, Simon-Chazottes D, Vandormael-Pournin S, Soueid J, Langa F, Cohen-Tannoudji M, Bernard BA, Panthier JJ - PLoS ONE (2012)

H2B-mCherry gene targeting by HR at the Dct locus.(A) Insertion of H2B-mCherry gene at the Dct locus. The DctI-SceI allele, the HR2 repair vector and the DctH2B-mCherry-Neo allele are represented from top to bottom. AvrII sites are indicated. There are no homologous sequences between DctI-SceI and HR2 close to the I-SceI site. A lightning denotes I-SceI expression from pCMV-I-SceI or pCAG-I-SceI plasmid. The 1.4 and 4.5 kb of Dct isogenic DNA are depicted by grey rectangles. (B) Southern blot analysis of Dct+/+, Dct I-SceI/+ES cells, and DctH2B-mCherry-Neo/+ ES clones 7E and 11E. ES clones 7E and 11E were obtained after transfection with the linear HR2 repair vector, and with both the supercoiled HR2 repair vector and pCAG-I-SceI expression plasmid, respectively. Genomic DNAs were digested with AvrII. The probe used for the hybridization is the external 5′ probe depicted by a black bar. The 3.9 kb fragment is carried by the Dct + and Dct I-SceI alleles, and the 6.5 kb fragment is distinctive of the Dct H2B-mCherry-Neo targeted allele.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383693&req=5

pone-0039895-g005: H2B-mCherry gene targeting by HR at the Dct locus.(A) Insertion of H2B-mCherry gene at the Dct locus. The DctI-SceI allele, the HR2 repair vector and the DctH2B-mCherry-Neo allele are represented from top to bottom. AvrII sites are indicated. There are no homologous sequences between DctI-SceI and HR2 close to the I-SceI site. A lightning denotes I-SceI expression from pCMV-I-SceI or pCAG-I-SceI plasmid. The 1.4 and 4.5 kb of Dct isogenic DNA are depicted by grey rectangles. (B) Southern blot analysis of Dct+/+, Dct I-SceI/+ES cells, and DctH2B-mCherry-Neo/+ ES clones 7E and 11E. ES clones 7E and 11E were obtained after transfection with the linear HR2 repair vector, and with both the supercoiled HR2 repair vector and pCAG-I-SceI expression plasmid, respectively. Genomic DNAs were digested with AvrII. The probe used for the hybridization is the external 5′ probe depicted by a black bar. The 3.9 kb fragment is carried by the Dct + and Dct I-SceI alleles, and the 6.5 kb fragment is distinctive of the Dct H2B-mCherry-Neo targeted allele.
Mentions: We thus decided to remove the two short regions of homology (including the loxP site) and to construct a novel repair vector. Therefore, a second destination vector (DV2) was produced using the H2B-mCherry reporter gene [31]. The HR2 repair vector was constructed (Fig. 2). It carries H2B-mCherry, a NeoR cassette flanked with FRT sites, two regions of homology with the DctI-SceI allele, 1.4 and 4.5 kb in length, and a HSV-TK negative selection cassette (Fig. 5A). By contrast with HR1, HR2 displays neither a short region of homology with the Dct gene next to the I-SceI site nor a loxP site.

Bottom Line: The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells.However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus.These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Mouse functional Genetics, Institut Pasteur, Paris, France.

ABSTRACT
Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

Show MeSH
Related in: MedlinePlus