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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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Production of a new target allele at the Dct locus.(A) Introduction of an I-SceI site at the Dct locus. From top to bottom are represented the Dct wild-type allele (Dct+), the replacement vector, the DctI-SceI-Neo targeted allele, and the DctI-SceI allele produced after deletion of the NeoR cassette. The grey boxes represent exons 1 and 2 of the Dct gene. The black circle represents 109 bp of Dct intron 1 sequence that are lost during an homologous recombination event. The horizontal black bar represents the external 5′ probe used for the Southern blots. The NeoR and HSV-TK cassettes are depicted as white rectangles. loxP sites are represented by white triangles. The Dct homologous arms, 1.9 and 4.5 kb in length, are denoted as grey rectangles. I-SceI and BamHI restriction sites are indicated. (B) Southern blot analysis of Dct+/+ ES cells and targeted ES cells (clone 4). Genomic DNAs of ES cells were digested with BamHI. The 11.7 and 6.4 kb fragments are distinctive of the Dct+ and DctI-SceI-Neo alleles, respectively. (C) Test of the ability of I-SceI meganuclease to specifically cleave DctI-SceI-Neo/+ ES cells. Southern blot analysis of Dct+/+ ES cells and clone 4. Genomic DNAs were digested with I-SceI and BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI-Neo allele. (D) Southern blot analysis of Dct+/+ ES cells, clone 4, MF1 and MF2 clones. Genomic DNAs were digested with BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI allele.
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pone-0039895-g003: Production of a new target allele at the Dct locus.(A) Introduction of an I-SceI site at the Dct locus. From top to bottom are represented the Dct wild-type allele (Dct+), the replacement vector, the DctI-SceI-Neo targeted allele, and the DctI-SceI allele produced after deletion of the NeoR cassette. The grey boxes represent exons 1 and 2 of the Dct gene. The black circle represents 109 bp of Dct intron 1 sequence that are lost during an homologous recombination event. The horizontal black bar represents the external 5′ probe used for the Southern blots. The NeoR and HSV-TK cassettes are depicted as white rectangles. loxP sites are represented by white triangles. The Dct homologous arms, 1.9 and 4.5 kb in length, are denoted as grey rectangles. I-SceI and BamHI restriction sites are indicated. (B) Southern blot analysis of Dct+/+ ES cells and targeted ES cells (clone 4). Genomic DNAs of ES cells were digested with BamHI. The 11.7 and 6.4 kb fragments are distinctive of the Dct+ and DctI-SceI-Neo alleles, respectively. (C) Test of the ability of I-SceI meganuclease to specifically cleave DctI-SceI-Neo/+ ES cells. Southern blot analysis of Dct+/+ ES cells and clone 4. Genomic DNAs were digested with I-SceI and BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI-Neo allele. (D) Southern blot analysis of Dct+/+ ES cells, clone 4, MF1 and MF2 clones. Genomic DNAs were digested with BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI allele.

Mentions: As a first step, the I-SceI restriction site was inserted within Dct intron 1 in ES cells. A replacement vector containing a unique I-SceI restriction site, a positive selection (NeoR) cassette flanked by loxP sites, and 1.9 and 4.5 kb of 5′ and 3′ genomic sequences from the Dct gene was constructed. A negative selection cassette (HSV-TK) was added after the 3′ homology arm (Fig. 3A). The replacement vector was linearized and electroporated into CK35 ES cells. The cells were cultured in the presence of G418 and gancyclovir. Out of 107 G418- and gancyclovir-resistant colonies, one clone (ES clone 4) was correctly targeted with the replacement vector as shown by PCR (data not shown), and later confirmed by Southern blot analysis (Fig. 3B). To test whether the meganuclease I-SceI is able to specifically cleave the new DctI-SceI-Neo allele, genomic DNA of the ES clone 4 was treated with both BamHI and I-SceI restriction enzymes. Southern blot analysis using an external 5′ probe revealed the 4.5 kb BamHI-I-SceI distinctive fragment, indicating that the I-SceI site inserted at the Dct locus was indeed cut in vitro by the meganuclease (Fig. 3C).


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)

Production of a new target allele at the Dct locus.(A) Introduction of an I-SceI site at the Dct locus. From top to bottom are represented the Dct wild-type allele (Dct+), the replacement vector, the DctI-SceI-Neo targeted allele, and the DctI-SceI allele produced after deletion of the NeoR cassette. The grey boxes represent exons 1 and 2 of the Dct gene. The black circle represents 109 bp of Dct intron 1 sequence that are lost during an homologous recombination event. The horizontal black bar represents the external 5′ probe used for the Southern blots. The NeoR and HSV-TK cassettes are depicted as white rectangles. loxP sites are represented by white triangles. The Dct homologous arms, 1.9 and 4.5 kb in length, are denoted as grey rectangles. I-SceI and BamHI restriction sites are indicated. (B) Southern blot analysis of Dct+/+ ES cells and targeted ES cells (clone 4). Genomic DNAs of ES cells were digested with BamHI. The 11.7 and 6.4 kb fragments are distinctive of the Dct+ and DctI-SceI-Neo alleles, respectively. (C) Test of the ability of I-SceI meganuclease to specifically cleave DctI-SceI-Neo/+ ES cells. Southern blot analysis of Dct+/+ ES cells and clone 4. Genomic DNAs were digested with I-SceI and BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI-Neo allele. (D) Southern blot analysis of Dct+/+ ES cells, clone 4, MF1 and MF2 clones. Genomic DNAs were digested with BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI allele.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383693&req=5

pone-0039895-g003: Production of a new target allele at the Dct locus.(A) Introduction of an I-SceI site at the Dct locus. From top to bottom are represented the Dct wild-type allele (Dct+), the replacement vector, the DctI-SceI-Neo targeted allele, and the DctI-SceI allele produced after deletion of the NeoR cassette. The grey boxes represent exons 1 and 2 of the Dct gene. The black circle represents 109 bp of Dct intron 1 sequence that are lost during an homologous recombination event. The horizontal black bar represents the external 5′ probe used for the Southern blots. The NeoR and HSV-TK cassettes are depicted as white rectangles. loxP sites are represented by white triangles. The Dct homologous arms, 1.9 and 4.5 kb in length, are denoted as grey rectangles. I-SceI and BamHI restriction sites are indicated. (B) Southern blot analysis of Dct+/+ ES cells and targeted ES cells (clone 4). Genomic DNAs of ES cells were digested with BamHI. The 11.7 and 6.4 kb fragments are distinctive of the Dct+ and DctI-SceI-Neo alleles, respectively. (C) Test of the ability of I-SceI meganuclease to specifically cleave DctI-SceI-Neo/+ ES cells. Southern blot analysis of Dct+/+ ES cells and clone 4. Genomic DNAs were digested with I-SceI and BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI-Neo allele. (D) Southern blot analysis of Dct+/+ ES cells, clone 4, MF1 and MF2 clones. Genomic DNAs were digested with BamHI. The 4.5 kb fragment is distinctive of the DctI-SceI allele.
Mentions: As a first step, the I-SceI restriction site was inserted within Dct intron 1 in ES cells. A replacement vector containing a unique I-SceI restriction site, a positive selection (NeoR) cassette flanked by loxP sites, and 1.9 and 4.5 kb of 5′ and 3′ genomic sequences from the Dct gene was constructed. A negative selection cassette (HSV-TK) was added after the 3′ homology arm (Fig. 3A). The replacement vector was linearized and electroporated into CK35 ES cells. The cells were cultured in the presence of G418 and gancyclovir. Out of 107 G418- and gancyclovir-resistant colonies, one clone (ES clone 4) was correctly targeted with the replacement vector as shown by PCR (data not shown), and later confirmed by Southern blot analysis (Fig. 3B). To test whether the meganuclease I-SceI is able to specifically cleave the new DctI-SceI-Neo allele, genomic DNA of the ES clone 4 was treated with both BamHI and I-SceI restriction enzymes. Southern blot analysis using an external 5′ probe revealed the 4.5 kb BamHI-I-SceI distinctive fragment, indicating that the I-SceI site inserted at the Dct locus was indeed cut in vitro by the meganuclease (Fig. 3C).

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