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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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Complementary ss gol-NLS-RecA-Gal4 filaments direct site-specific insertion of a gene trap into the gol locus. (A) Two regions of the gol gene corresponding to exons 4 through 5 (gol-270, 270 bp) and exon 6 (gol-300) were amplified, denatured and coated with NLS-RecA-Gal4 protein to make complementary ss-gol-NLS-RecA-Gal4 filaments. Filaments were injected with the EGFP gene trap that contains a SA followed by EGFP in three reading frames and a poly adenylation signal (pA). (B) PCR amplification of junction fragments between the EGFP gene trap and the endogenous gol locus was observed with DNA isolated from individual embryos. Only primers proximal to the region complementary to the filament yielded amplification products that could be verified by sequencing (asterisks). (C) Junction fragments allow insertions to be mapped to the gol locus. The gol-270 filaments (black bar) resulted in insertion of the EGFP gene trap 5′ and 3′ to the region complementary to the filaments (black triangles). In some cases, identical junction fragments were observed from different embryos (triangles marked 4×). The triangle marked 4x corresponds to the 3′-end of the gol-270 filament. The gol-300 filament (clear bar) also promoted insertion near regions 5′ to the complementary sequence in the gol gene (clear triangles).
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Figure 3: Complementary ss gol-NLS-RecA-Gal4 filaments direct site-specific insertion of a gene trap into the gol locus. (A) Two regions of the gol gene corresponding to exons 4 through 5 (gol-270, 270 bp) and exon 6 (gol-300) were amplified, denatured and coated with NLS-RecA-Gal4 protein to make complementary ss-gol-NLS-RecA-Gal4 filaments. Filaments were injected with the EGFP gene trap that contains a SA followed by EGFP in three reading frames and a poly adenylation signal (pA). (B) PCR amplification of junction fragments between the EGFP gene trap and the endogenous gol locus was observed with DNA isolated from individual embryos. Only primers proximal to the region complementary to the filament yielded amplification products that could be verified by sequencing (asterisks). (C) Junction fragments allow insertions to be mapped to the gol locus. The gol-270 filaments (black bar) resulted in insertion of the EGFP gene trap 5′ and 3′ to the region complementary to the filaments (black triangles). In some cases, identical junction fragments were observed from different embryos (triangles marked 4×). The triangle marked 4x corresponds to the 3′-end of the gol-270 filament. The gol-300 filament (clear bar) also promoted insertion near regions 5′ to the complementary sequence in the gol gene (clear triangles).

Mentions: We examined whether the EGFP gene trap could be detected in the gol gene in genomic DNA isolated from embryos following co-injection of either css-gol-270-NLS-RecA-Gal4 or css-gol-300-NLS-RecA-Gal4 filaments. Because these filaments target different regions in the same gene, we could test whether insertion of the EGFP reporter gene clustered around the genomic sequence in the co-injected filament. Following co-injection of either filament with the EGFP gene trap, embryos displaying EGFP fluorescence were selected for DNA isolation and PCR analysis. To analyze potential junction fragments between the reporter gene trap and the gol gene, primers were designed to PCR amplify between the reporter gene trap (Primer III, Figure 3A) and regions of the gol gene proximal to the position corresponding to the filaments (Primer I or Primer II, Figure 3A). PCR on genomic DNA isolated from embryos co-injected with the reporter gene trap and css-gol-270-NLS-RecA-Gal4 (primer Pairs I and III) led to amplification of several PCR products (Figure 3B, left panel). Similar PCR results with multiple products were observed after amplification using primer Pair II and III on DNA isolated from embryos co-injected with the reporter gene trap and css-gol-300-NLS-RecA-Gal4 filaments. Sequencing of these products revealed that they represented junction fragments between the reporter gene trap and the gol gene. Insertion sites were observed both 5′ and 3′ to the region complementary to the css-gol-270-NLS-RecA-Gal4 filament (Figure 3C black triangles, and Supplementary Table S4). Remarkably, the same junction fragment that corresponds to the 3′-end of the css-gol-270-NLS-RecA-Gal4 filament was recovered independently in four different embryos, and an additional embryo produced a junction fragment that differed by only one nucleotide. Regions 5′ to the css-gol-270-NLS-RecA-Gal4 filament also had common insertion site. Amplification with primer Pairs I and III was specific to the injection from the css-gol-270-NLS-RecA-Gal4 filament and did not amplify products from DNA isolated from css-gol-300-NLS-RecA-Gal4 filament-injected embryos, with the exception of one embryo that produced false amplification products as revealed by sequencing. The same was true for primer Pairs II and III, which only amplified PCR products from DNA isolated from embryos co-injected with the reporter gene trap and the css-gol-300-NLS-RecA-Gal4 filaments. Analysis of these PCR products revealed junction fragments that sit 5′ to the css-gol-300-NLS-RecA-Gal4 filaments sequence (Figure 3C, white arrows). While the degree of off-target integration was not assessed in these experiments, our results indicate that the NLS-RecA-Gal4 filaments direct integration of the EGFP reporter gene to regions near the complementary genomic location of the filament.Figure 3.


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

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

Complementary ss gol-NLS-RecA-Gal4 filaments direct site-specific insertion of a gene trap into the gol locus. (A) Two regions of the gol gene corresponding to exons 4 through 5 (gol-270, 270 bp) and exon 6 (gol-300) were amplified, denatured and coated with NLS-RecA-Gal4 protein to make complementary ss-gol-NLS-RecA-Gal4 filaments. Filaments were injected with the EGFP gene trap that contains a SA followed by EGFP in three reading frames and a poly adenylation signal (pA). (B) PCR amplification of junction fragments between the EGFP gene trap and the endogenous gol locus was observed with DNA isolated from individual embryos. Only primers proximal to the region complementary to the filament yielded amplification products that could be verified by sequencing (asterisks). (C) Junction fragments allow insertions to be mapped to the gol locus. The gol-270 filaments (black bar) resulted in insertion of the EGFP gene trap 5′ and 3′ to the region complementary to the filaments (black triangles). In some cases, identical junction fragments were observed from different embryos (triangles marked 4×). The triangle marked 4x corresponds to the 3′-end of the gol-270 filament. The gol-300 filament (clear bar) also promoted insertion near regions 5′ to the complementary sequence in the gol gene (clear triangles).
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Figure 3: Complementary ss gol-NLS-RecA-Gal4 filaments direct site-specific insertion of a gene trap into the gol locus. (A) Two regions of the gol gene corresponding to exons 4 through 5 (gol-270, 270 bp) and exon 6 (gol-300) were amplified, denatured and coated with NLS-RecA-Gal4 protein to make complementary ss-gol-NLS-RecA-Gal4 filaments. Filaments were injected with the EGFP gene trap that contains a SA followed by EGFP in three reading frames and a poly adenylation signal (pA). (B) PCR amplification of junction fragments between the EGFP gene trap and the endogenous gol locus was observed with DNA isolated from individual embryos. Only primers proximal to the region complementary to the filament yielded amplification products that could be verified by sequencing (asterisks). (C) Junction fragments allow insertions to be mapped to the gol locus. The gol-270 filaments (black bar) resulted in insertion of the EGFP gene trap 5′ and 3′ to the region complementary to the filaments (black triangles). In some cases, identical junction fragments were observed from different embryos (triangles marked 4×). The triangle marked 4x corresponds to the 3′-end of the gol-270 filament. The gol-300 filament (clear bar) also promoted insertion near regions 5′ to the complementary sequence in the gol gene (clear triangles).
Mentions: We examined whether the EGFP gene trap could be detected in the gol gene in genomic DNA isolated from embryos following co-injection of either css-gol-270-NLS-RecA-Gal4 or css-gol-300-NLS-RecA-Gal4 filaments. Because these filaments target different regions in the same gene, we could test whether insertion of the EGFP reporter gene clustered around the genomic sequence in the co-injected filament. Following co-injection of either filament with the EGFP gene trap, embryos displaying EGFP fluorescence were selected for DNA isolation and PCR analysis. To analyze potential junction fragments between the reporter gene trap and the gol gene, primers were designed to PCR amplify between the reporter gene trap (Primer III, Figure 3A) and regions of the gol gene proximal to the position corresponding to the filaments (Primer I or Primer II, Figure 3A). PCR on genomic DNA isolated from embryos co-injected with the reporter gene trap and css-gol-270-NLS-RecA-Gal4 (primer Pairs I and III) led to amplification of several PCR products (Figure 3B, left panel). Similar PCR results with multiple products were observed after amplification using primer Pair II and III on DNA isolated from embryos co-injected with the reporter gene trap and css-gol-300-NLS-RecA-Gal4 filaments. Sequencing of these products revealed that they represented junction fragments between the reporter gene trap and the gol gene. Insertion sites were observed both 5′ and 3′ to the region complementary to the css-gol-270-NLS-RecA-Gal4 filament (Figure 3C black triangles, and Supplementary Table S4). Remarkably, the same junction fragment that corresponds to the 3′-end of the css-gol-270-NLS-RecA-Gal4 filament was recovered independently in four different embryos, and an additional embryo produced a junction fragment that differed by only one nucleotide. Regions 5′ to the css-gol-270-NLS-RecA-Gal4 filament also had common insertion site. Amplification with primer Pairs I and III was specific to the injection from the css-gol-270-NLS-RecA-Gal4 filament and did not amplify products from DNA isolated from css-gol-300-NLS-RecA-Gal4 filament-injected embryos, with the exception of one embryo that produced false amplification products as revealed by sequencing. The same was true for primer Pairs II and III, which only amplified PCR products from DNA isolated from embryos co-injected with the reporter gene trap and the css-gol-300-NLS-RecA-Gal4 filaments. Analysis of these PCR products revealed junction fragments that sit 5′ to the css-gol-300-NLS-RecA-Gal4 filaments sequence (Figure 3C, white arrows). While the degree of off-target integration was not assessed in these experiments, our results indicate that the NLS-RecA-Gal4 filaments direct integration of the EGFP reporter gene to regions near the complementary genomic location of the filament.Figure 3.

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