PUBLICATION
Highly efficient germ-line transmission of proviral insertions in zebrafish
- Authors
- Gaiano, N., Allende, M., Amsterdam, A., Kawakami, K., and Hopkins, N.
- ID
- ZDB-PUB-970326-7
- Date
- 1996
- Source
- Proceedings of the National Academy of Sciences of the United States of America 93(15): 7777-7782 (Journal)
- Registered Authors
- Allende, Miguel L., Amsterdam, Adam, Gaiano, Nicholas, Hopkins, Nancy, Kawakami, Koichi
- Keywords
- none
- MeSH Terms
-
- Genome, Viral
- Cell Line
- Peptide Elongation Factors/genetics
- Recombinant Proteins/biosynthesis
- Vesicular stomatitis Indiana virus/genetics*
- Moloney murine leukemia virus/genetics*
- Transfection/methods*
- Peptide Elongation Factor 1
- Promoter Regions, Genetic
- Viral Envelope Proteins/biosynthesis
- Viral Envelope Proteins/genetics
- Microinjections
- Proviruses/genetics*
- Zebrafish
- Animals
- Molecular Sequence Data
- beta-Galactosidase/biosynthesis
- DNA Primers
- Polymerase Chain Reaction
- Animals, Genetically Modified
- 3T3 Cells
- Base Sequence
- Repetitive Sequences, Nucleic Acid
- Mice
- Xenopus
- Genes, Reporter
- PubMed
- 8755552 Full text @ Proc. Natl. Acad. Sci. USA
Citation
Gaiano, N., Allende, M., Amsterdam, A., Kawakami, K., and Hopkins, N. (1996) Highly efficient germ-line transmission of proviral insertions in zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 93(15):7777-7782.
Abstract
An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping