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Use of RecA fusion proteins to induce genomic modifications in zebrafish.

Liao HK, Essner JJ - Nucleic Acids Res. (2011)

Bottom Line: Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos.Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations.Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA-DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

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Related in: MedlinePlus

Targeted mutations of the gol locus using gol-NLS-RecA-Gal4 filaments were transmitted through the zebrafish germline. (A and B). A testcross between a F0 zebrafish injected with gol-NLS-RecA-Gal4 filaments and a golb1 homozygote produces offspring that fail to complement the golb1 allele (B) and siblings with normal pigment (A).
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Figure 4: Targeted mutations of the gol locus using gol-NLS-RecA-Gal4 filaments were transmitted through the zebrafish germline. (A and B). A testcross between a F0 zebrafish injected with gol-NLS-RecA-Gal4 filaments and a golb1 homozygote produces offspring that fail to complement the golb1 allele (B) and siblings with normal pigment (A).

Mentions: Embryos displaying EGFP expression in the eye after co-injection of css-gol-DNA-NLS-RecA-Gal4 filaments with the reporter gene trap into wild-type embryos were grown to adulthood and screened for transmission of targeted alleles in the gol gene to the next generation. The adult F0 zebrafish were tested for non-complementation with the golb1 allele by crossing to homozygous golb1 fish. Out of 21 F0 adults screened, two F0 fish produced offspring that failed to complement the golb1 allele (Figure 4A and Table 3), which corresponds to 9.5% of the injected F0 zebrafish. The germlines of these two founder fish are mosaic, with 0.7 and 3.7% of the offspring showing failure to complement the golb1 allele (Table 3). One of the F0 founders was injected with css-gol-270-NLS-RecA-Gal4 filaments and the other with css-gol-300-NLS-RecA-Gal4 filaments (Figure 3). None of the offspring from either founder displayed fluorescence from the EGFP gene trap. This suggested that the new mutant alleles of gol were not insertion alleles of the EGFP gene trap, but could represent deletions or rearrangements.Figure 4.


Use of RecA fusion proteins to induce genomic modifications in zebrafish.

Liao HK, Essner JJ - Nucleic Acids Res. (2011)

Targeted mutations of the gol locus using gol-NLS-RecA-Gal4 filaments were transmitted through the zebrafish germline. (A and B). A testcross between a F0 zebrafish injected with gol-NLS-RecA-Gal4 filaments and a golb1 homozygote produces offspring that fail to complement the golb1 allele (B) and siblings with normal pigment (A).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105420&req=5

Figure 4: Targeted mutations of the gol locus using gol-NLS-RecA-Gal4 filaments were transmitted through the zebrafish germline. (A and B). A testcross between a F0 zebrafish injected with gol-NLS-RecA-Gal4 filaments and a golb1 homozygote produces offspring that fail to complement the golb1 allele (B) and siblings with normal pigment (A).
Mentions: Embryos displaying EGFP expression in the eye after co-injection of css-gol-DNA-NLS-RecA-Gal4 filaments with the reporter gene trap into wild-type embryos were grown to adulthood and screened for transmission of targeted alleles in the gol gene to the next generation. The adult F0 zebrafish were tested for non-complementation with the golb1 allele by crossing to homozygous golb1 fish. Out of 21 F0 adults screened, two F0 fish produced offspring that failed to complement the golb1 allele (Figure 4A and Table 3), which corresponds to 9.5% of the injected F0 zebrafish. The germlines of these two founder fish are mosaic, with 0.7 and 3.7% of the offspring showing failure to complement the golb1 allele (Table 3). One of the F0 founders was injected with css-gol-270-NLS-RecA-Gal4 filaments and the other with css-gol-300-NLS-RecA-Gal4 filaments (Figure 3). None of the offspring from either founder displayed fluorescence from the EGFP gene trap. This suggested that the new mutant alleles of gol were not insertion alleles of the EGFP gene trap, but could represent deletions or rearrangements.Figure 4.

Bottom Line: Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos.Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations.Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA-DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

Show MeSH
Related in: MedlinePlus