Highly efficient germ-line transmission of proviral insertions in zebrafish.
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.
Gaiano
N
N
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Allende
M
M
Amsterdam
A
A
Kawakami
K
K
Hopkins
N
N
D016475:3T3 Cells; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D017930:Genes, Reporter; D016679:Genome, Viral; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection / Q000379:methods*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics; D014981:Xenopus; D015027:Zebrafish; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D001616:beta-Galactosidase