Molecular Biology and Genetics of Viruses
Malcolm J. Fraser, Jr.
Rev. Julius A. Nieuwland, CSC, Professor of Biological Sciences
Ph.D., Ohio State University
Postdoctoral, Penn State University, Texas A & M University
Fellow, American Association for the Advancement of Science
Fellow, Royal Entomological Society
Fellow, Entomological Society of America
Fellow, American Academy of Microbiology
The Fraser laboratory merges research in Molecular Virology and Transgenic Engineering with the particular goals of advancing applications that improve the human condition. A major thrust of the research in this laboratory concerns the utilization of molecular approaches to understanding and manipulating virus genetics in ways that permit beneficial transgenic alteration of the invertebrate hosts of these viruses.
The Home of piggyBac
The Fraser lab has a solid history in the molecular genetics of Baculoviruses, both from the standpoint of their exploitation as expression vectors and as an experimental system for the isolation and analysis of a unique family of Lepidopteran transposons. Our laboratory is responsible for the characterization and development of the piggyBac transposon (http://piggybac.bio.nd.edu), a highly versatile transposon vector with wide utility for transgenic engineering in a host of eukaryotic species. This transposon is now facilitating applications of genetic manipulation and gene characterization for a wide range of important invertebrate species that previously had few genetic tools. Important invertebrate species including the economically significant silkworm, Bombyx mori , and human disease vectors including Aedes aegypti and Anopheles stephensi , may now be transgenically engineered with relative facility, allowing the analysis of gene identity, expression, and function.
PiggyBac has also found utility in mammalian genetic engineering including mouse, rat and porcine animal models, as well as human cells. Most recently, piggyBac has been used in the establishment and differentiation of induced pluripotent stem cells, an important and viable alternative to embryonic stem cells.
Research Emphasis in Molecular Virology
The Molecular Virology aspects of our research include exploring genetic strategies for suppressing vectored virus infections in mosquito cells including the viral disease agents for Dengue Fever, Yellow Fever, West Nile Fever and Chikungunya virus. These are among the most devastating disease agents of humankind. Hepatitis C and HIV are widespread devastating chronic human disease viruses for which a cure has remained elusive. Our approach to inhibiting all of these viruses involves the use of catalytic RNA molecules called Ribozymes. The success of this strategy for antiviral suppression has been remarkable, and may eventually lead to eradication of vectored alphaviruses or flaviruses, and cures for Hepatitis C and HIV/AIDS.
Research Emphasis in Transgenic Engineering
The Invertebrate Transgenesis aspects of our research effort involve applications in significant Lepidopteran and Dipteran insects. Among Lepidopteran insects, we seek to improve caterpillars as bioreactors for the production of human therapeutic gene products. This involves various transgenic approaches that serve to alter properties of the insect systems that limit its utility for these purposes, and enhance those properties that are attractive. Research in this direction involves a significant effort to uncover strategies for optimization and controlled expression of genes in these insect systems.
Among the Dipteran insects, our principal effort involves the development of strategies for transgenic modification of mosquito vectors to reduce their capacity for vectoring human disease agents, particularly Flavivirus disease agents like Dengue Fever Virus. While transposon vectors such as piggyBac remain a significant tool in these transgenesis applications, our lab also explores alternative approaches to effecting transgenesis of these mosquito vectors to provide a versatile “toolbox” for functional genomics in these important disease arthropods. While Drosophila melanogaster is not a focus of our research it remains a significant model genetic system that we utilize to validate our transgenesis approaches prior to testing them in other insect systems.
We also have a vigorous program of research in transgenic silkworms. Silkworms are natural protein biocreators and may be engineered to produce recombinant human diagnostic and therapeutic proteins, as well as recombinant silks. One recent application has been the development of recombinant spider silk production in transgenic silkworms (see: www.kraiglabs.com).
Research Emphasis in Induced Pluripotent Stem Cells
We are taking advantage of piggyBac’s utility in reprogramming cells for pluripotency to develop induced pluripotent stem cell strategies for several neurological disorders. Of principal concern is the Neimann-Pick C type 1 and 2, for which we are using mouse model systems to attempt to develop induced pluripotent stem cell therapies.
- U.S. Patent No. 4,870,023. "Recombinant baculovirus occlusion bodies in vaccines and biological control." M. J. Fraser, E. Rosen, and V. Ploplis.American Biogenetics Sciences, Inc.
- U.S. Patent No. 5,041,379. "Heliothis expression systems." M. J. Fraser, E. Rosen, and V. Ploplis. American Biogenetics Sciences, Inc.
- U. S. Patent No. 6,218,185. “The piggyBac Transposon for Transgenic Engineering of Insects.” M. J. Fraser, P. D. Shirk, T. A. Elick, and O. P. Perera.USDA/ARS, University of Florida, and University of Notre Dame.
- U. S. Patent No. 6,551,825. “piggyBac transposon-based genetic transformation system for insects.” P. D. Shirk, M. J. Fraser, T. A. Elick and O. P. Perera. USDA/ARS, University of Florida and University of Notre Dame.
- U. S. Patent No. 6,962,810. “Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggyBac.” M. J. Fraser, X. Li. University of Notre Dame.
- U. S. Patent No. 7,105,343. “Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggyBac.” M. J. Fraser, X. Li. University of Notre Dame.
- U. S. Patent No. 7,932,088.. “High efficiency transformation of Plasmodium falciparum by the Lepidopteran transposon piggyBac.” J. H. Adams, M. J. Fraser, B. Balu, and D. A. Shoue. University of Notre Dame.
M.J. Fraser Jr., L. Carey, K. Boonvisudhi, and H.G.H. Wang. (1995). “Assay for movement of Lepidepteran transposon IFP2 in insect cells using a Baculovirus genome as a target DNA”. Virology 211:397-407.
A.M. Handler, S.D. McCombs, M.J. Fraser, S.J. Saul. (1998). “The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly”. Proc. Natl. Acad. Sci. USA 95:7520-7525.
T. Tamura, C. Thibert, C. Royer, T. Karda, E. Abraham, M. Kamba, N. Komoto, J. Thomas, B. Mauchamp, G. Chavancy, P. Shirk, M.J. Fraser, J. Prudhomme, and P. Couble. (2000). “Germline transformation of the pilleworm Bombyx mori L. using a piggyBac transposon-derived vector”. Nat. Biotech, 18:81-84.
N. Lobo, A. Hua-Van, X. Li, B. M. Nolan and M. J. Fraser. (2002). “Germ live transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBc vector”. Ins. Mol. Biol., 11:133-139.
A. Sarkar, C. Sim, Y. S. Hong, J. R. Hogan, M. J. Fraser, H. M. Robertson, F. H. Collins. (2003). “Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences”. Mol. Gen. Genomics 270:173-180.
X. Li, R. Harrell, A. Handler, T. Beam, K. Hennessy, and M.J. Fraser. (2004). “piggyBac internal sequences are necessary for efficient transformation of target genomes”. Insect Mol. Biol. 14:17-30
B. Balu, D. A. Shoue, M. J. Fraser and J. Adams. (2005). “High efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac “. Proc. Natl. Acad. Sci., 102:16391-6.
N. Lobo, T.S. Fraser, J. Adams, and M.J. Fraser. (2006). “Interplasmid transposition demonstrates piggyBac mobility in vertebrate species”. Genetica, 128:347-57.
E.T. Shinohara, J.M. Kaminski, D.J. Segal, P. Pelczar, R. Kolhe, T. Ryan, C.J. Coates, M.J. Fraser, A.M. Handler, R. Yanagimachi, and S. Moisyadi. (2007). “Active integration: new strategies for transgenesis”. Transgenic Res. 16:333-339.
X. Shi, R.L. Harrison, J.R. Hollister, A. Mohammed, M.J. Fraser Jr, and D.L. Jarvis. (2007). “Construction and characterization of new piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and the identification of a previously unrecognized activator sequence in piggyBac”. BMC Biotechnol., 7:5-23.
N. Sethurama, M.J. Fraser, Jr., P. Eggleston, and D A. O’Brochta. (2007). “Post-integration stability of piggyBac in Aedes aegypti”. Insect. Biochem. Mol. Biol. 37:941-951.
J.H. Keith, T.S. Fraser, and M.J. Fraser, Jr. 2008. “Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues”. BMC Molec. Biol. 9:72.
J. Keith, C. Schaeper, T. S. Fraser, and M. J. Fraser, Jr. (2008). “Mutational analysis of highly conserved acidic amino acids in the piggyBac transposase”. BMC Mol. Biol. 9:73.
J. Chompoosri, T. Fraser, Y. Rongsriyam, N. Komalamisra, P. Siriyasatien, U. Thavara, A. Tawatsin, and M. J. Fraser, Jr. (2009). “Intramolecular integration assay validates integrase Phi C31 and R4 potential in a variety of insect cells”. Southeast Asian J. Trop. Med. Public Health. 40:1235-1254.
B. Balu, C. Chauhan, S.P. Maher, D. A. Shoue, H. Wang, J. Kissinger, M.J. Fraser Jr. and J.H. Adams. (2009). “Whole-genome mutagenesis of Plasmodium falciparum identifies critical blood-stage genes”. BMC Microbiology, 9:83.
P. Nawtaisong, J. Keith, T. Fraser, V.Balaraman, A. Kolokoltsov, R. Davey, S. Higgs, A. Mohammed, Y. Rongsriyam, N. Komolamisra, M.J. Fraser, Jr.: (2009) “Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome”. Virology Journal, 6:73. PMID:19497123
A.G. Lynch, F. Tanzer, M.J. Fraser, E.G. Shephard, A.L. Williamson, E.P. Rybicki. (2010). “Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production”. BMC Biotechnol.10:30.
H.J. Ferguson, L.G. Neven, S.T. Thibault, A. Mohammed, M.J. Fraser. (2010). “Genetic transformation of the codling moth, Cydia pomonella L., with piggyBac EGFP”. Transgenic Res. [Epub ahead of print]
J.R. Carter, J.H. Keith, P.V. Barde, T.S. Fraser, & M.J. Fraser, Jr. (2010) “Targeting of highly conserved Dengue virus sequences with anti-Dengue virustrans-splicing group I introns”. BMC Mol Biol, 11:84. PMID:21078188