Several biotechnology companies in the Boston area have beaten a pathto the door of molecular biologist Nancy Hopkins, who raises andmutates zebrafish. "What they are thinking about," Hopkins toldBioWorld Today, "is developing a company, away from my laboratory,to use zebrafish as a vertebrate gene-finding device."Hopkins isn't sure that the idea makes sense. She would rather "build afacility in the Boston area, jointly between the Massachusetts Instituteof Technology (MIT) and Massachusetts General Hospital (MGH),with biotech company participation. It would do large-scale gene-screens, based on insertional mutation of the zebrafish."Best known in pet stores, Danio rerio, the zebrafish, is coming up fastas an in vivo model for studying vertebrate development and geneticanalysis. What does this minnow-size, smartly striped fish have thatlaboratory mice don't have?For one thing, fast fecundity: A female zebrafish lays 400 eggs a week,or 20,800 a year, each 0.7 millimeters in diameter. Embryos hatch in48 hours. From egg to breeding adult takes about 11 weeks.Zebrafish's Clear AdvantageA mother mouse (Mus musculus), admittedly closer to Homo sapienson the evolutionary scale (100 million years apart) than zebrafish (400million), gestates her clutch of six to 12 embryos for about three weeks,and they mature reproductively four weeks after birth.Above all, living zebrafish embryos are as transparent as raindrops,giving scientists a clear window for following, and perturbing,development of single cells in vivo.Where mice are one-up on the fish is their aptitude for mutating toorder, and forming transgenic animals. Hopkins, and zebrafish researchteams on two continents, are trying to educate zebrafish to accept andexpress foreign genes of interest, and permit random insertionalmutations in their own genome.This week's Science carries a paper by Hopkins and her group titled,"Integration and Germ-Line Transmission of a Pseudotype RetroviralVector in Zebrafish."The "pseudotyped" viral construct they describe is a first-round stab ata retroviral vector that will infect the genome of a fish. It consists of aMoloney mouse leukemia virus (MoMLV) payload enclosed in avesicular stomatitis virus (VSV) subunit nose-cone. This cell-penetrating, genome-entering package was developed for human genetherapy by molecular geneticist Theo Friedmann, a co-author of thepaper, at the University of California at San Diego.Hopkins explained: "This pseudovirus has a very broad host range. Itmust be that its receptor shares with many types of cells some commoncomponent on the cell membrane. VSV, which penetrates themembrane, is a viciously lytic virus, and kills what it infects. But co-infected with MoMLV," she continued, "you get benign mixedparticles, with expanded host range. Some people, she said, are evenusing it to infect mosquitos."To determine if this mixed viral vehicle could infect zebrafishembryos, especially their developing germ-line cells, the team injectedthe vector into zebrafish embryos at the blastula stage (2,000 to 4,000cells), three or four hours after fertilization.Of 51 embryos grown to maturity, eight transmitted the proviral DNAto their progeny, which in turn passed on the viral message to 50percent of their third-generation offspring, in true Mendelianproportion.Improving on the pseudotype's efficiency is Hopkins' first goal."Because it was designed to work in humans and mice, it turns out thatthe particular promoter used on this virus doesn't work in fish," shesaid.Seeking A Fish-Specific PromoterHopkins' colleague, Shuo Lin, is first author on the Science paper. "Wehave already made some retroviral vectors by ourselves, with differentconstructs," he told BioWorld Today, "in order to get better expression.Then over the next six months or so we will make a large number oftransfected zebrafish _ maybe 200 _ to determine if this retroviralintegration can cause mutations, and at what frequency."We have to have something like a fish-specific promoter," Lin added."Once it works in zebrafish, it can work in other fish too."On which note, Hopkins observed, "In commercial fisheries, I thinkpeople interested in getting their fish to grow faster will take an interestin fish made transgenic for recombinant growth hormone.""By chemical mutagenesis," she said, "you can do enormous-scalesaturation mutagenesis screens, and get a defect in virtually everysingle gene of the zebrafish genome."Such a mutation causes a detectable change in a single embryo. "If youcould do it by insertional mutagenesis, instead of chemically," she said,"then you could rapidly clone the gene, and study defects in zebrafish'sdeveloping organs, such as the pancreas, thymus or heart."That's precisely what cardiologist Mark Fishman is doing at MGH inBoston, where he directs the Cardiac Research Center. "We'veconcentrated on the heart," he told BioWorld Today, "because I'm acardiologist, and organ development is particularly suitable to study inzebrafish. For one thing, it's a vertebrate, so its organs are relevant tohumans. For another, it has a transparent embryo, which _ unlikealmost every other embryo _ is accessible, manipulable, andvizualizable for studying cardiac development."If and when Hopkins goes forward with the biotech companies' idea ofsetting up a zebrafish "vertebrate gene finder" consortium, jointly withMIT and MGH, Fishman would be the MGH end of the venture, shesaid."This is still very premature," he said.But Fishman did sum up Hopkins' contribution to the burgeoningzebrafish research field:"Nancy Hopkins' general work has been to investigate better methodsfor inserting genes into the genome of the fish. That is importantbecause, in theory, a certain percentage of the time, when exogenousDNA is incorporated into the genome, it will cause a mutation. Whenyou cause a mutation that way, you are able to use the exogenous,marked, sequence as a tag to pull out that region, and the surroundingregion of DNA."That means it is usually more straightforward for identifying the genethat is mutated than if you use a chemical mutagen _ the standard way_ which doesn't carry a tag with it."The difficulty in the zebrafish field has been designing efficientmethods for doing this. Many labs have been working on that, with thesame goal: To develop an efficient method for transgenesis, which isadding genes, or insertional mutagenesis, which means interruptinggenes." n
-- David N. Leff Science Editor
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