Regulation of retroviral gene expression was the dominant research interest from 1980-1995. We used Rous sarcoma virus (RSV) as a model for these studies. The questions we addressed were: 1) How is unspliced retroviral RNA sorted for packaging and translation? 2) What are the roles of the three short open reading frames in the 5' leader sequence of RSV RNA? 3) Is overexpression of pp60v-src gene expression cytotoxic? Beginning with Bob Petersen (Professor in Pathology at Case Western Reserve University in Cleveland, OH Chuck Hensel (Myriad Genetics, Salt Lake City, UT), and Todd Smith (President of Geospiza, Inc. in Seattle, WA) and continuing with Aris Moustakas (Ludwig Institute for Cancer Research in Uppsula, Sweden,), and Tad Sonstegard (USDA Gene Evaluation & Mapping Laboratory in Beltsville, MD), the answers were found to the first questions. Essentially, the Gag protein or its polyprotein precursor autogenously sorts full-length RSV RNA into pools for translation and packaging and the open reading frames appear to assist ribosomes (or 40S ribosomal subunits) traverse the highly structured 5' leader sequence of the RSV mRNAs. The open reading frames are translated and their positions and lengths are highly conserved within a conserved RNA secondary structure that is common for all avian leukosis and sarcoma viruses. New methods for mapping ribosome binding-sites, causing DNA-mediated virus transformation, studying translational regulation (and the critical size of ORFs beyond which efficient reinitiation does not occur as well as the subtle effects of alteration of RNA secondary structure) were produced. Li-Wha Wu (a Professor at the National Cheng Kung University Hospital in Tainan, Taiwan) showed that overexpression of pp60v-src was indeed cytotoxic and that the levels of pp60v-src within transformed cells was close to the threshold for morbidity. More importantly, she demonstrated how cells in tissue culture could adapt to residual expression of an oncogene like v-src and thereby change their phenotype. During the early phases of this work, a concurrent project on mouse ribosomal protein L32 was conducted by Pam Mulligan and Colleen Jacks (Head of the Biology Dept. at Gustavus Adolphus College in St. Peter, MN), who isolated and characterized the expression of a processed copy, L32', of the most active gene. The role of L32' is unknown however, the gene has what looks to be a fully active CAAT/TATA promoter normally not seen in ribosomal protein genes.
In 1986, our lab began collaboration with Profs. Anne Kapuscinski (Dept. of Fisheries and Wildlife), Kevin Guise (Dept. of Animal Sciences; deceased 1991), and Tony Faras (Dept. of Genetics, Cell Biology and Development) to make growth-enhanced transgenic fish (northern pike, walleye, trout). This was the beginning to the Minnesota Superfish Story. There were groups in Minnesota that wanted bigger fish for sport fishing and aquaculture. Growth enhancement was defined as either 1) achieving adult larger size, 2) faster growth rates, 3) more efficient feed conversion, or 4) some combination of the first three. We microinjected hundreds of thousands of eggs from walleye, northern pike, salmon, and trout. Some of the fish grew faster and larger. Zhanjiang Liu (now a Professor in the Fisheries Dept. at Auburn University in Alabama) isolated the carp b-actin gene, defined its transcriptional regulatory elements, and used both it and those from Rous sarcoma virus and SV40 to make expression vectors to drive expression of growth hormone genes. Liu worked closely with visitors to the lab, Professors Zuoyan Zhu (Head of the Institute of Hydrobiology in Wuhan, China) and Boaz Moav (Dept. of Zoology at Tel Aviv University), as well as an undergraduate partner, Kevin Roberg (now at Cal Tech). Because big fish only spawn once or twice a year, we needed an alternative source for fish embryos to measure transgene expression in fish. For this reason we brought zebrafish into the lab. By 1990, we had several lines of transgenic fish containing exogenous growth hormone genes. But, rather than being able to put them in outdoor facilities for inexpensive fish rearing, the Minnesota Environmental Quality Board instituted a set of rules that effectively quashed the superfish project. By 1996, all of our large transgenic fish had been sacrificed, although Scott Fahrenkrug (see below) continued a collaboration with Prof. Jo-Ann Leong in Oregon, to make transgenic vectors for immunizing rainbow trout against Infectious Hematopoietic Necrosis Virus.
Bringing zebrafish into the lab opened up new areas of research. Spearheaded by Jeff Essner (now at the Huntsman Cancer Institute at the University of Utah in Salt Lake City), zebrafish became our model system for examining the regulation of gene expression during embryonic development. Jeff focused on the zebrafish thyroid hormone receptor (c-erbA) genes and also followed up a serendipitous cloning of the zebrafish connexin 43.5 gene. Jeff's connexin work led to a collaboration with our next door neighbor, Prof. Ross Johnson, where Carla Finis began, and Alison Krufka continues characterizing other zebrafish connexin genes. Scott Fahrenkrug (USDA Anima Research Lab in Clay Center, NE) conducted a project that linked our interests in translational regulation in RSV infected cells to translational regulation of normal vertebrate mRNAs by examining the expression of translational initiation factor eIF-4E during zebrafish development. [Jeff, Scott, and Russ Essner designed and built with the help of others our first zebrafish facility, which was replaced by our new fish room run by Angie Lanie.] Karl Clark followed in Scott's footsteps for awhile by looking for internal ribosome entry sequences in natural mRNAs that may be important to growth and development of zebrafish. In collaboration with Karl, Geri Kantor is examining effects on translation of the 5' leader sequence of the zebrafish c-erbA mRNAs, which have a complexity that rivals that of the RSV mRNAs. Chris Kaufman pursued a project that linked our interests in early development with enhanced fish growth and normal development. Chris characterized the expression of the zebrafish c-ski genes, which were initially identified by Gonzalo Martinez (CSIC, Spain; a Fullbright fellow) and which encode factors that affect muscle development as well as early dorsal-ventral axis formation. Chris also worked on several aspects of transposition by the Sleeping Beauty transposase (see below). Mark Foreman brought his own project to the lab, a continuation of that he began in Michigan, an analysis of tubulin gene expression during eye regeneration. All of these projects involved analysis of expression of exogenous transgenes during fish development. We thought that making transgenic zebrafish would be fast, easy, and cheap. It isn't.
Liu and Boaz had shown us that it was difficult to make transgenic fish, even zebrafish, with reproducible levels of gene expression. As a result, beginning with Ling He (Bethesda, MD), who characterized retrotransposon sequences in zebrafish, several methods for accelerating early integration were examined. Soon thereafter, Luba Caldovic looked for ways to protect transgenes against "position effects," the term used to describe the finding that the level of expression of a transgene often is dependent upon where it integrates in an animal's genome. Luba demonstrated that "border elements", special sequences that flank genes in the genome and protect them from being regulated by the enhancers and silencers of other genes, from Drosophila and chicken genomes could be used in zebrafish to eliminate position effects. Ling's work to enhance integration frequencies was followed by that of Zsuzsa Izsvak and Zoltan Ivics (both now are at the Max Delbruck Center for Molecular Medicine in Berlin, Germany) who began very serious efforts to enhance the rates of transgene integration into zebrafish genomes. They first showed that retroviral integrase could accelerate integration and increase expression of transgenic DNA. Reasoning that endogenous recombinases would be the best way to achieve early integration of transgenes, they followed up this work with investigations of retroposons and transposons in zebrafish genomes. They characterized many repetitive elements and showed how these elements can be used to screen the zebrafish genome for polymorphisms. The culmination of their work was the resurrection of the Sleeping Beauty (SB) transposase gene that led to the development of the first vertebrate transposon system. Deanna Mohn (now at the Mount Sinai School of Medicine Deanna) demonstrated that the transposon system could be used as a vector for transfer of genes into zebrafish genomes. Further development of the Sleeping Beauty System is a major focus of our lab. Karl Clark developed a series of gene-trap vectors based on Fluorescent Protein expression as markers for temporal and tissue specificity of gene activity of the tagged locus. Geyi Liu, with help from Geri Kantor, is investigating several aspects of the specificity of the transposition reaction. Aron Guerts is participating in development of a more efficient mutagenesis system by looking for SB-expressing zebrafish, as well as making a series of constructs that will be used in collaborations with Geyi and Geri to determine size-effects of transposons and methods to enhance transposition rates. We also have established several collaborations with colleagues in the Department of Genetics, Cell Biology and Development that led to the funding of the Beckman Center for Transposon Research. The P.I.s of the Beckman Center are Stephen Ekker, David Largaespada and Scott McIvor. We are collaborating with other colleagues, Cliff Steer and Betsy Kren as well as Scott McIvor and Chet Whitley to investigate the potential for using the SB system for human gene therapy. As part of this endeavor, Cheryl Linehan-Stieers is developing methods for high efficiency transfer of the transposons into mammalian liver cells.
A new line of research, initiated by Dritan Agalliu and Chris Kaufman (see above), is to develop an efficient method for gene inactivation in zebrafish without using stem cells. Zongbin Cui is investigating the potential of RecA-type proteins to direct homologous recombination in zebrafish.