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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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Mapping of new gol alleles from Founders 1, 4, 6 and 7 with z-markers reveals deletions at the gol locus. Founders were crossed with golb1 homozygotes and DNA isolated from offspring displaying the golb1 phenotype and from siblings (sib) with wild-type pigmentation. Parental lines (P0 and golb1) or the wild-type strain used for injection (WIK) is shown for comparison. DNA was amplified with primer pairs to identify SSLPs that were polymorphic. (A) Offspring from Founder 1 that fail to complement the golb1 mutation have one allele for markers SSLPs z46013, golin8, z9404 and z8488, but are heterozygous for marker z21330. This indicates a deletion of 32.5–38.5 CM, with a proximal breakpoint boundary that maps between z8488 and z21330. (B) In offspring from Founder 4 the only SSLP alleles present were from the golb1 parent, indicating the deletion is >38.5 CM. (C) In the offspring from Founder 6 that failed to complement the golb1 mutation, a single allele was present for marker z9404 but heterozygous for marker z21330, indicating a deletion of at least 7 CM. (D) Founder 7 produced offspring with a deletion that contained only marker z9404, indicating a deletion of at least 7 CM.
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Figure 5: Mapping of new gol alleles from Founders 1, 4, 6 and 7 with z-markers reveals deletions at the gol locus. Founders were crossed with golb1 homozygotes and DNA isolated from offspring displaying the golb1 phenotype and from siblings (sib) with wild-type pigmentation. Parental lines (P0 and golb1) or the wild-type strain used for injection (WIK) is shown for comparison. DNA was amplified with primer pairs to identify SSLPs that were polymorphic. (A) Offspring from Founder 1 that fail to complement the golb1 mutation have one allele for markers SSLPs z46013, golin8, z9404 and z8488, but are heterozygous for marker z21330. This indicates a deletion of 32.5–38.5 CM, with a proximal breakpoint boundary that maps between z8488 and z21330. (B) In offspring from Founder 4 the only SSLP alleles present were from the golb1 parent, indicating the deletion is >38.5 CM. (C) In the offspring from Founder 6 that failed to complement the golb1 mutation, a single allele was present for marker z9404 but heterozygous for marker z21330, indicating a deletion of at least 7 CM. (D) Founder 7 produced offspring with a deletion that contained only marker z9404, indicating a deletion of at least 7 CM.

Mentions: To determine if the new gol alleles correspond to deletions, we used polymorphic z-markers or PCR-amplified simple SSLPs to examine the genotypes of the F1 embryos around the gol locus on chromosome 18. In total, 22 SSLPs or z-markers were tested to identify polymorphic loci for Founders 1, 4, 6 and 7 for which we could identify the parental and test cross genotypes. Polymorphic loci were not identified for Founders 2, 3 and 5. For Founder 1, we were able to identify polymorphic amplification products between the wild-type strain used for injection experiments and the AB strain that harbors the golb1 allele using z-markers z46013, z9404, z8488 and z21330 (Figure 5A and Table 3). Marker z46013 is close to the telomere and the gol locus, while z9404, z8488 and z21330 are ∼7, 32.5 and 38.5 CM away from the telomere, respectively (http:/zfin.org). Founder 1 produced offspring from the test cross that lacked the z46013, z9404 and z8488 markers and retained one of the z21330 markers from the wild-type-injected parent. This suggests the deletion proximal breakpoint maps between z8488 and z21330 and is 32.5 CM to 38.5 CM in size. We also identified a polymorphic marker, called golin8, within intron 8 of the gol gene that was also lost from the wild-type background (Figure 5A), further confirming the deletion of the gol locus in embryos derived from Founder 1.Figure 5.


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

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

Mapping of new gol alleles from Founders 1, 4, 6 and 7 with z-markers reveals deletions at the gol locus. Founders were crossed with golb1 homozygotes and DNA isolated from offspring displaying the golb1 phenotype and from siblings (sib) with wild-type pigmentation. Parental lines (P0 and golb1) or the wild-type strain used for injection (WIK) is shown for comparison. DNA was amplified with primer pairs to identify SSLPs that were polymorphic. (A) Offspring from Founder 1 that fail to complement the golb1 mutation have one allele for markers SSLPs z46013, golin8, z9404 and z8488, but are heterozygous for marker z21330. This indicates a deletion of 32.5–38.5 CM, with a proximal breakpoint boundary that maps between z8488 and z21330. (B) In offspring from Founder 4 the only SSLP alleles present were from the golb1 parent, indicating the deletion is >38.5 CM. (C) In the offspring from Founder 6 that failed to complement the golb1 mutation, a single allele was present for marker z9404 but heterozygous for marker z21330, indicating a deletion of at least 7 CM. (D) Founder 7 produced offspring with a deletion that contained only marker z9404, indicating a deletion of at least 7 CM.
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Related In: Results  -  Collection

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Figure 5: Mapping of new gol alleles from Founders 1, 4, 6 and 7 with z-markers reveals deletions at the gol locus. Founders were crossed with golb1 homozygotes and DNA isolated from offspring displaying the golb1 phenotype and from siblings (sib) with wild-type pigmentation. Parental lines (P0 and golb1) or the wild-type strain used for injection (WIK) is shown for comparison. DNA was amplified with primer pairs to identify SSLPs that were polymorphic. (A) Offspring from Founder 1 that fail to complement the golb1 mutation have one allele for markers SSLPs z46013, golin8, z9404 and z8488, but are heterozygous for marker z21330. This indicates a deletion of 32.5–38.5 CM, with a proximal breakpoint boundary that maps between z8488 and z21330. (B) In offspring from Founder 4 the only SSLP alleles present were from the golb1 parent, indicating the deletion is >38.5 CM. (C) In the offspring from Founder 6 that failed to complement the golb1 mutation, a single allele was present for marker z9404 but heterozygous for marker z21330, indicating a deletion of at least 7 CM. (D) Founder 7 produced offspring with a deletion that contained only marker z9404, indicating a deletion of at least 7 CM.
Mentions: To determine if the new gol alleles correspond to deletions, we used polymorphic z-markers or PCR-amplified simple SSLPs to examine the genotypes of the F1 embryos around the gol locus on chromosome 18. In total, 22 SSLPs or z-markers were tested to identify polymorphic loci for Founders 1, 4, 6 and 7 for which we could identify the parental and test cross genotypes. Polymorphic loci were not identified for Founders 2, 3 and 5. For Founder 1, we were able to identify polymorphic amplification products between the wild-type strain used for injection experiments and the AB strain that harbors the golb1 allele using z-markers z46013, z9404, z8488 and z21330 (Figure 5A and Table 3). Marker z46013 is close to the telomere and the gol locus, while z9404, z8488 and z21330 are ∼7, 32.5 and 38.5 CM away from the telomere, respectively (http:/zfin.org). Founder 1 produced offspring from the test cross that lacked the z46013, z9404 and z8488 markers and retained one of the z21330 markers from the wild-type-injected parent. This suggests the deletion proximal breakpoint maps between z8488 and z21330 and is 32.5 CM to 38.5 CM in size. We also identified a polymorphic marker, called golin8, within intron 8 of the gol gene that was also lost from the wild-type background (Figure 5A), further confirming the deletion of the gol locus in embryos derived from Founder 1.Figure 5.

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