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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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Expression of an EGFP gene trap is consistent with site-specific integration into the gol, flh and prom1a loci. Single-stranded NLS-RecA-Gal4 filaments complementary to regions of the gol (A), flh (B) and prom1a (C) genes were co-injected with the EGFP gene trap cassettes. EGFP expression consistent with targeting gene expression was observed in 5–19% of the injected embryos (Table 2). For the gol gene, expression was observed in the retina at 3 dpf (white arrows, A), for the flh gene expression was detected in the notochord at 14 hpf (arrows, B; arrowheads mark the notochord boundary) and for the prom1a gene expression was detected in the dorsal diencephalon at 30 hpf (arrows, C).
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Figure 2: Expression of an EGFP gene trap is consistent with site-specific integration into the gol, flh and prom1a loci. Single-stranded NLS-RecA-Gal4 filaments complementary to regions of the gol (A), flh (B) and prom1a (C) genes were co-injected with the EGFP gene trap cassettes. EGFP expression consistent with targeting gene expression was observed in 5–19% of the injected embryos (Table 2). For the gol gene, expression was observed in the retina at 3 dpf (white arrows, A), for the flh gene expression was detected in the notochord at 14 hpf (arrows, B; arrowheads mark the notochord boundary) and for the prom1a gene expression was detected in the dorsal diencephalon at 30 hpf (arrows, C).

Mentions: The data presented above indicated that the css-gol-NLS-RecA-Gal4 filaments are able to promote LOH at the gol locus. Our analysis of DNA from the mutated embryos indicated that the disrupted gol allele was not caused by gene replacement, resulting in mutation of the locus. However, LOH could be a result of DNA DSB, including chromosomal loss, targeted deletions or rearrangements of the gol locus. To test whether DSBs might contribute to the LOH, we co-injected a gene trap that contains an EGFP reporter gene and the css-gol-NLS-RecA-Gal4 filaments. We reasoned if the css-gol-NLS-RecA-Gal4 filaments were promoting site-specific DSBs at the gol locus, at some frequency we would observe integration of the gene trap during the repair of the locus by the non-homologous end-joining (NHEJ) pathway. The NHEJ repair pathway is the predominant pathway in the early zebrafish embryo (24–26). The reporter/gene trap used in this assay is a linear dsDNA construct that contains a SA from the carp β-actin gene followed by EGFP coding sequence that lacks the first AUG (27). These sequences are followed by the zebrafish β-actin 3′-UTR/PolyA signal (pA). The EGFP gene trap was placed in all three reading frames following the SA (Supplementary Table S3). Integration of the EGFP gene trap in an intron is expected to produce a fluorescent fusion protein from the targeted gene in one of six integrations (three reading frames and two possible orientations). The detected fluorescence signal is expected to correlate with the endogenous expression of the targeted gene if correct targeting is accomplished. A css-gol-270-NLS-RecA-Gal4 filament complementary to 270 bp of the gol locus spanning exons 4 and 5 was co-injected with linear dsDNA for all three frames of the EGFP gene trap into wild-type embryos. Following injection expression of EGFP was observed in the retinal epithelium of the eye where the gol gene is normally expressed in 5.1% of the embryos (Figure 2A and Table 2). Similar results were obtained with a second filament, css-gol-300-NLS-RecA-Gal4 that has a sequence complementary to 300 bp spanning introns 5 to 6 of the gol gene. In three experiments, expression of EGFP was observed in the eyes in 8.5% of the embryos (Table 2). For each filament EGFP expression in regions of the embryo outside the eye was only observed in a few embryos, suggesting effective targeting by the NLS-RecA-Gal4 filaments to the gol locus. The results suggest that filaments produced from two different regions of the gol gene are able to direct integration into the gol locus.Figure 2.


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

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

Expression of an EGFP gene trap is consistent with site-specific integration into the gol, flh and prom1a loci. Single-stranded NLS-RecA-Gal4 filaments complementary to regions of the gol (A), flh (B) and prom1a (C) genes were co-injected with the EGFP gene trap cassettes. EGFP expression consistent with targeting gene expression was observed in 5–19% of the injected embryos (Table 2). For the gol gene, expression was observed in the retina at 3 dpf (white arrows, A), for the flh gene expression was detected in the notochord at 14 hpf (arrows, B; arrowheads mark the notochord boundary) and for the prom1a gene expression was detected in the dorsal diencephalon at 30 hpf (arrows, C).
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Related In: Results  -  Collection

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Figure 2: Expression of an EGFP gene trap is consistent with site-specific integration into the gol, flh and prom1a loci. Single-stranded NLS-RecA-Gal4 filaments complementary to regions of the gol (A), flh (B) and prom1a (C) genes were co-injected with the EGFP gene trap cassettes. EGFP expression consistent with targeting gene expression was observed in 5–19% of the injected embryos (Table 2). For the gol gene, expression was observed in the retina at 3 dpf (white arrows, A), for the flh gene expression was detected in the notochord at 14 hpf (arrows, B; arrowheads mark the notochord boundary) and for the prom1a gene expression was detected in the dorsal diencephalon at 30 hpf (arrows, C).
Mentions: The data presented above indicated that the css-gol-NLS-RecA-Gal4 filaments are able to promote LOH at the gol locus. Our analysis of DNA from the mutated embryos indicated that the disrupted gol allele was not caused by gene replacement, resulting in mutation of the locus. However, LOH could be a result of DNA DSB, including chromosomal loss, targeted deletions or rearrangements of the gol locus. To test whether DSBs might contribute to the LOH, we co-injected a gene trap that contains an EGFP reporter gene and the css-gol-NLS-RecA-Gal4 filaments. We reasoned if the css-gol-NLS-RecA-Gal4 filaments were promoting site-specific DSBs at the gol locus, at some frequency we would observe integration of the gene trap during the repair of the locus by the non-homologous end-joining (NHEJ) pathway. The NHEJ repair pathway is the predominant pathway in the early zebrafish embryo (24–26). The reporter/gene trap used in this assay is a linear dsDNA construct that contains a SA from the carp β-actin gene followed by EGFP coding sequence that lacks the first AUG (27). These sequences are followed by the zebrafish β-actin 3′-UTR/PolyA signal (pA). The EGFP gene trap was placed in all three reading frames following the SA (Supplementary Table S3). Integration of the EGFP gene trap in an intron is expected to produce a fluorescent fusion protein from the targeted gene in one of six integrations (three reading frames and two possible orientations). The detected fluorescence signal is expected to correlate with the endogenous expression of the targeted gene if correct targeting is accomplished. A css-gol-270-NLS-RecA-Gal4 filament complementary to 270 bp of the gol locus spanning exons 4 and 5 was co-injected with linear dsDNA for all three frames of the EGFP gene trap into wild-type embryos. Following injection expression of EGFP was observed in the retinal epithelium of the eye where the gol gene is normally expressed in 5.1% of the embryos (Figure 2A and Table 2). Similar results were obtained with a second filament, css-gol-300-NLS-RecA-Gal4 that has a sequence complementary to 300 bp spanning introns 5 to 6 of the gol gene. In three experiments, expression of EGFP was observed in the eyes in 8.5% of the embryos (Table 2). For each filament EGFP expression in regions of the embryo outside the eye was only observed in a few embryos, suggesting effective targeting by the NLS-RecA-Gal4 filaments to the gol locus. The results suggest that filaments produced from two different regions of the gol gene are able to direct integration into the gol locus.Figure 2.

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