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High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish

By Wenbiao Chen, Shawn Burgess, Greg Golling, Adam Amsterdam and Nancy Hopkins


Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate A major task for biologists in the postgenome era is to determine the cellular and organismal functions of an estimated 30,000 human genes (24, 49). A powerful approach for functional determination is forward genetic screens in model organisms. By altering genes at random with mutagens and screening for abnormalities caused by the mutations, genetic screens have yielded large collections of mutants with defects in development, physiology, and/or behavior, and many o

Year: 2001
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