ZFIN ID: ZDB-PUB-110131-26
Use of RecA fusion proteins to induce genomic modifications in zebrafish
Liao, H.K., and Essner, J.J.
Date: 2011
Source: Nucleic acids research   39(10): 4166-4179 (Journal)
Registered Authors: Essner, Jeffrey
Keywords: none
MeSH Terms:
  • Animals
  • DNA, Single-Stranded/administration & dosage
  • DNA-Binding Proteins/genetics
  • Embryo, Nonmammalian/anatomy & histology
  • Eye Color
  • Gene Targeting
  • Genes, Reporter
  • Genome
  • Injections
  • Loss of Heterozygosity*
  • Mutation
  • Nuclear Localization Signals
  • Rec A Recombinases/genetics
  • Rec A Recombinases/metabolism*
  • Recombinant Fusion Proteins/administration & dosage
  • Recombinant Fusion Proteins/chemistry
  • Zebrafish/anatomy & histology
  • Zebrafish/embryology
  • Zebrafish/genetics*
PubMed: 21266475 Full text @ Nucleic Acids Res.
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.
ADDITIONAL INFORMATION