PUBLICATION
Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin
- Authors
- Wu, Y., Zhang, G., Xiong, Q., Luo, F., Cui, C., Hu, W., Yu, Y., Su, J., Xu, A., and Zhu, Z.
- ID
- ZDB-PUB-060313-4
- Date
- 2006
- Source
- Marine biotechnology (New York, N.Y.) 8(3): 304-311 (Journal)
- Registered Authors
- Hu, Wei
- Keywords
- GH gene, integration, knockin, knockout, zebrafish
- MeSH Terms
-
- Animals
- Gene Deletion*
- Gene Expression Regulation
- Genome/genetics
- Green Fluorescent Proteins/genetics*
- Larva
- Mutagenesis, Site-Directed/methods*
- Organisms, Genetically Modified/genetics*
- Zebrafish/genetics*
- PubMed
- 16501876 Full text @ Mar. Biotechnol.
Citation
Wu, Y., Zhang, G., Xiong, Q., Luo, F., Cui, C., Hu, W., Yu, Y., Su, J., Xu, A., and Zhu, Z. (2006) Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin. Marine biotechnology (New York, N.Y.). 8(3):304-311.
Abstract
Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F(1) generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P(0) generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping