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Genetic manipulation of schistosomes – progress with integration competent vectors

  • SUTAS SUTTIPRAPA (a1), GABRIEL RINALDI (a1) (a2) and PAUL J. BRINDLEY (a1)

Summary

Draft genome sequences for Schistosoma japonicum and S. mansoni are now available. The schistosome genome encodes ∼13 000 protein-encoding genes for which the functions of few are well understood. Nonetheless, the new genes represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past 15 years, noteworthy progress has been achieved towards development of tools for gene manipulation and transgenesis of schistosomes. A brief history of genetic manipulation is presented, along with a review of the field with emphasis on reports of integration of transgenes into schistosome chromosomes.

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Corresponding author

*Corresponding author: Paul J. Brindley, Department of Microbiology, Immunology & Tropical Medicine, George Washington University Medical Center, Ross Hall, 2300 I Street, NW, Washington, DC 20037USA. Fax: +1 202 994 2913. E-mail: paul.brindley@gwumc.edu

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Adjalley, S. H., Lee, M. C. and Fidock, D. A. (2010). A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. Methods in Molecular Biology 634, 87100.
Alrefaei, Y. N., Okatcha, T. I., Skinner, D. E. and Brindley, P. J. (2011). Progress with schistosome transgenesis. Memórias do Instituto Oswaldo Cruz (in press).
Ayuk, M. A., Suttiprapa, S., Rinaldi, G., Mann, V. H., Lee, C. M. and Brindley, P. J. (2011). Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules. International Journal for Parasitology 41, 783789.
Balu, B., Shoue, D. A., Fraser, M. J. Jr. and Adams, J. H. (2005). High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac. Proceedings of the National Academy of Sciences, USA 102, 1639116396.
Beckmann, S., Wippersteg, V., El-Bahay, A., Hirzmann, J., Oliveira, G. and Grevelding, C. G. (2007). Schistosoma mansoni: germ-line transformation approaches and actin-promoter analysis. Experimental Parasitology 117, 292303.
Berriman, M., Haas, B. J., LoVerde, P. T., Wilson, R. A., Dillon, G. P., Cerqueira, G. C., Mashiyama, S. T., Al-Lazikani, B., Andrade, L. F., Ashton, P. D., Aslett, M. A., Bartholomeu, D. C., Blandin, G., Caffrey, C. R., Coghlan, A., Coulson, R., Day, T. A., Delcher, A., DeMarco, R., Djikeng, A., Eyre, T., Gamble, J. A., Ghedin, E., Gu, Y., Hertz-Fowler, C., Hirai, H., Hirai, Y., Houston, R., Ivens, A., Johnston, D. A., Lacerda, D., Macedo, C. D., McVeigh, P., Ning, Z., Oliveira, G., Overington, J. P., Parkhill, J., Pertea, M., Pierce, R. J., Protasio, A. V., Quail, M. A., Rajandream, M. A., Rogers, J., Sajid, M., Salzberg, S. L., Stanke, M., Tivey, A. R., White, O., Williams, D. L., Wortman, J., Wu, W., Zamanian, M., Zerlotini, A., Fraser-Liggett, C. M., Barrell, B. G. and El-Sayed, N. M. (2009). The genome of the blood fluke Schistosoma mansoni. Nature 460, 352358.
Bischof, J. and Basler, K. (2008). Recombinases and their use in gene activation, gene inactivation, and transgenesis. Methods in Molecular Biology 420, 175195.
Boutros, M. and Ahringer, J. (2008). The art and design of genetic screens: RNA interference. Nature Reviews Genetics 9, 554566.
Boyle, J. P., Wu, X. J., Shoemaker, C. B. and Yoshino, T. P. (2003). Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts. Molecular and Biochemical Parasitology 128, 205215.
Brindley, P. J., Mitreva, M., Ghedin, E. and Lustigman, S. (2009). Helminth genomics: the implications for human health. PLoS Neglected Tropical Diseases 3: e538.
Brindley, P. J. and Pearce, E. J. (2007). Genetic manipulation of schistosomes. International Journal for Parasitology 37, 465473.
Buguliskis, J. S., Brossier, F., Shuman, J. and Sibley, L. D. (2010). Rhomboid 4 (ROM4) affects the processing of surface adhesins and facilitates host cell invasion by Toxoplasma gondii. PLoS Pathogens 6: e1000858.
Burns, J. C., Friedmann, T., Driever, W., Burrascano, M. and Yee, J. K. (1993). Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proceedings of the National Academy of Sciences, USA 90, 80338037.
Cary, L. C., Goebel, M., Corsaro, B. G., Wang, H. G., Rosen, E. and Fraser, M. J. (1989). Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172, 156169.
Correnti, J. M., Brindley, P. J. and Pearce, E. J. (2005). Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth. Molecular and Biochemical Parasitology 143, 209215.
Correnti, J. M., Jung, E., Freitas, T. C. and Pearce, E. J. (2007). Transfection of Schistosoma mansoni by electroporation and the description of a new promoter sequence for transgene expression. International Journal for Parasitology 37, 11071115.
Correnti, J. M. and Pearce, E. J. (2004). Transgene expression in Schistosoma mansoni: introduction of RNA into schistosomula by electroporation. Molecular and Biochemical Parasitology 137, 7579.
Coustau, C. and Yoshino, T. P. (2000). Flukes without snails: advances in the in vitro cultivation of intramolluscan stages of trematodes. Experimental Parasitology 94, 6266.
Couto, L. B. and High, K. A. (2010). Viral vector-mediated RNA interference. Current Opinion in Pharmacology 10, 534542.
Damasceno, J. D., Beverley, S. M. and Tosi, L. R. (2010). A transposon toolkit for gene transfer and mutagenesis in protozoan parasites. Genetica 138, 301311.
Davis, R. E., Parra, A., LoVerde, P. T., Ribeiro, E., Glorioso, G. and Hodgson, S. (1999). Transient expression of DNA and RNA in parasitic helminths by using particle bombardment. Proceedings of the National Academy of Sciences, USA 96, 86878692.
Dirks, C. and Miller, A. D. (2001). Many nonmammalian cells exhibit postentry blocks to transduction by gammaretroviruses pseudotyped with various viral envelopes, including vesicular stomatitis virus G glycoprotein. Journal of Virology 75, 63756383.
Dvorak, J., Beckmann, S., Lim, K. C., Engel, J. C., Grevelding, C. G., McKerrow, J. H. and Caffrey, C. R. (2010). Biolistic transformation of Schistosoma mansoni: Studies with modified reporter-gene constructs containing regulatory regions of protease genes. Molecular and Biochemical Parasitology 170, 3740.
Elick, T. A., Bauser, C. A. and Fraser, M. J. (1996). Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase. Genetica 98, 3341.
Emi, N., Friedmann, T. and Yee, J. K. (1991). Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus. Journal of Virology 65, 12021207.
Faghiri, Z. and Skelly, P. J. (2009). The role of tegumental aquaporin from the human parasitic worm, Schistosoma mansoni, in osmoregulation and drug uptake. FASEB Journal 23, 27802789.
Feschotte, C. and Pritham, E. J. (2007). DNA transposons and the evolution of eukaryotic genomes. Annual Review of Genetics 41, 331368.
Fraser, M. J., Brusca, J. S., Smith, G. E. and Summers, M. D. (1985). Transposon-mediated mutagenesis of a baculovirus. Virology 145, 356361.
Fraser, M. J., Ciszczon, T., Elick, T. and Bauser, C. (1996). Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Molecular Biology 5, 141151.
Freitas, T. C., Jung, E. and Pearce, E. J. (2007). TGF-beta signaling controls embryo development in the parasitic flatworm Schistosoma mansoni. PLoS pathogens 3: e52.
Gilbert, C., Schaack, S., Pace, J. K. 2nd, Brindley, P. J. and Feschotte, C. (2010). A role for host-parasite interactions in the horizontal transfer of transposons across phyla. Nature 464, 13471350.
Giordano-Santini, R. and Dupuy, D. (2011). Selectable genetic markers for nematode transgenesis. Cellular and Molecular Life Sciences 68, 19171927.
Gonzalez-Estevez, C., Momose, T., Gehring, W. J. and Salo, E. (2003). Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker. Proceedings of the National Academy of Sciences, USA 100, 1404614051.
Grevelding, C. G. (2006). Transgenic flatworms. In Parasitic Flatworms. Molecular Biology, Biochemisrty, Immunolgy and Physiology (eds. Maule, A. G., and Marks, N. J.), pp. 149173. CABI, Wallingford, UK.
Han, Z. G., Brindley, P. J., Wang, S. Y. and Chen, Z. (2009). Schistosoma genomics: new perspectives on schistosome biology and host-parasite interaction. Annual Review of Genomics and Human Genetics 10, 211240.
Hannon, G. J. and Rossi, J. J. (2004). Unlocking the potential of the human genome with RNA interference. Nature 431, 371378.
Heyers, O., Walduck, A. K., Brindley, P. J., Bleiss, W., Lucius, R., Dorbic, T., Wittig, B. and Kalinna, B. H. (2003). Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts. Experimental Parasitology 105, 174178.
Hoffmann, K. F., Johnston, D. A. and Dunne, D. W. (2002). Identification of Schistosoma mansoni-gender associated transcripts by cDNA microarray profiling. Genome Biology 3: research0041-research0041.12
Homann, O. R., Dea, J., Noble, S. M. and Johnson, A. D. (2009). A phenotypic profile of the Candida albicans regulatory network. PLoS Genetics 5: e1000783.
Hotez, P. J., Brindley, P. J., Bethony, J. M., King, C. H., Pearce, E. J. and Jacobson, J. (2008). Helminth infections: the great neglected tropical diseases. Journal of Clinical Investigation 118, 13111321.
Ivics, Z., Li, M. A., Mates, L., Boeke, J. D., Nagy, A., Bradley, A. and Izsvak, Z. (2009). Transposon-mediated genome manipulation in vertebrates. Nature Methods 6, 415422.
Jurberg, A. D., Goncalves, T., Costa, T. A., de Mattos, A. C., Pascarelli, B. M., de Manso, P. P., Ribeiro-Alves, M., Pelajo-Machado, M., Peralta, J. M., Coelho, P. M. and Lenzi, H. L. (2009). The embryonic development of Schistosoma mansoni eggs: proposal for a new staging system. Development Genes and Evolution 219, 219234.
Kalinna, B. H. and Brindley, P. J. (2007). Manipulating the manipulators: advances in parasitic helminth transgenesis and RNAi. Trends in Parasitology 23, 197204.
Kapp, K., Coustau, C., Wippersteg, V., Jourdane, J., Kunz, W. and Grevelding, C. G. (2003). Transplantation of in vitro-generated Schistosoma mansoni mother sporocysts into Biomphalaria glabrata. Parasitology Research 91, 482485.
Kines, K. J., Mann, V. H., Morales, M. E., Shelby, B. D., Kalinna, B. H., Gobert, G. N., Chirgwin, S. R. and Brindley, P. J. (2006). Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus. Experimental Parasitology 112, 209220.
Kines, K. J., Morales, M. E., Mann, V. H., Gobert, G. N. and Brindley, P. J. (2008). Integration of reporter transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus. FASEB Journal 22, 29362948.
Kines, K. J., Rinaldi, G., Okatcha, T. I., Morales, M. E., Mann, V. H., Tort, J. F. and Brindley, P. J. (2010). Electroporation facilitates introduction of reporter transgenes and virions into schistosome eggs. PLoS Neglected Tropical Diseases 4: e593.
Krautz-Peterson, G., Bhardwaj, R., Faghiri, Z., Tararam, C. A. and Skelly, P. J. (2010). RNA interference in schistosomes: machinery and methodology. Parasitology 137, 485495.
Kumagai, T., Osada, Y., Ohta, N. and Kanazawa, T. (2009). Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide. Molecular and Biochemical Parasitology 164, 2631.
Lambeth, L. S., Moore, R. J., Muralitharan, M., Dalrymple, B. P., McWilliam, S. and Doran, T. J. (2005). Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs. BMC Biotechnology 5, 13.
Langridge, G. C., Phan, M. D., Turner, D. J., Perkins, T. T., Parts, L., Haase, J., Charles, I., Maskell, D. J., Peters, S. E., Dougan, G., Wain, J., Parkhill, J. and Turner, A. K. (2009). Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Research 19, 23082316.
Lewis, F. A. (1998). Schistosomiasis. Current Protocols in Immunology. Suppl. 28. In Animal Models for Infectious Diseases (eds. Coligan, J. E., Kruisbeek, A., M., Margulies, D. H., Shevach, E. M., and Strober, W.), Wiley, New York.
Linford, A. S., Moreno, H., Good, K. R., Zhang, H., Singh, U. and Petri, W. A. Jr. (2009). Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica. BMC Microbiology 9, 38.
Manjunath, N. and Dykxhoorn, D. M. (2010). Advances in synthetic siRNA delivery. Discovery Medicine 9, 418430.
Mann, V. H., Morales, M. E., Kines, K. J. and Brindley, P. J. (2008). Transgenesis of schistosomes: approaches employing mobile genetic elements. Parasitology 135, 141153.
Mann, V. H., Morales, M. E., Rinaldi, G. and Brindley, P. J. (2010). Culture for genetic manipulation of developmental stages of Schistosoma mansoni. Parasitology 137, 451462.
Mann, V. H., Suttiprapa, S., Rinaldi, G. and Brindley, P. J. (2011). Establishing transgenic schistosomes. PLoS Neglected Tropical Diseases, (in press).
Mastromarino, P., Conti, C., Goldoni, P., Hauttecoeur, B. and Orsi, N. (1987). Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH. Journal of General Virology 68, 23592369.
Mates, L., Izsvak, Z. and Ivics, Z. (2007). Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives. Genome Biology, 8 (Suppl 1), S1.
Miller, A. D. (1992). Human gene therapy comes of age. Nature 357, 455460.
Morales, M. E., Mann, V. H., Kines, K. J., Gobert, G. N., Fraser, M. J. Jr., Kalinna, B. H., Correnti, J. M., Pearce, E. J. and Brindley, P. J. (2007). piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni. FASEB Journal 21, 34793489.
Morales, M. E., Rinaldi, G., Gobert, G. N., Kines, K. J., Tort, J. F. and Brindley, P. J. (2008). RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade. Molecular and Biochemical Parasitology 157, 160168.
Mourão, M. M., Dinguirard, N., Franco, G. R., Yoshino, T. P. (2009). Phenotypic screen of early-developing larvae of the blood fluke, Schistosoma mansoni, using RNA interference. PLoS Neglected Tropical Diseases 3: e502.
O'Neill, M. T., Phuong, T., Healer, J., Richard, D. and Cowman, A. F. (2011). Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: implications for applied site-specific recombination. International Journal for Parasitology 41, 117123.
Petrus, I., Chuah, M. and VandenDriessche, T. (2010). Gene therapy strategies for hemophilia: benefits versus risks. Journal of Gene Medicine 12, 797809.
Plasterk, R. H., Izsvak, Z. and Ivics, Z. (1999). Resident aliens: the Tc1/mariner superfamily of transposable elements. Trends in Genetics 15, 326332.
Rinaldi, G., Morales, M. E., Alrefaei, Y. N., Cancela, M., Castillo, E., Dalton, J. P., Tort, J. F. and Brindley, P. J. (2009). RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs. Molecular and Biochemical Parasitology 167, 118126.
Rinaldi, G., Morales, M. E., Cancela, M., Castillo, E., Brindley, P. J. and Tort, J. F. (2008). Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica. PLoS Neglected Tropical Diseases 2: e260.
Rinaldi, G., Suttiprapa, S. and Brindley, P. J. (2011). Quantitative retrotransposon anchored PCR confirms transduction efficiency of transgenes in adult Schistosoma mansoni. Molecular and Biochemical Parasitology 177, 7076.
Rossi, A., Wippersteg, V., Klinkert, M. Q. and Grevelding, C. G. (2003). Cloning of 5′ and 3′ flanking regions of the Schistosoma mansoni calcineurin A gene and their characterization in transiently transformed parasites. Molecular and Biochemical Parasitology 130, 133138.
Sayed, A. A., Cook, S. K. and Williams, D. L. (2006). Redox balance mechanisms in Schistosoma mansoni rely on peroxiredoxins and albumin and implicate peroxiredoxins as novel drug targets. Journal of Biological Chemistry 281, 1700117010.
Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium (2009). The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature 460, 345351.
Semple, J. I., Garcia-Verdugo, R. and Lehner, B. (2010). Rapid selection of transgenic C. elegans using antibiotic resistance. Nature Methods 7, 725727.
Shibata, N., Rouhana, L. and Agata, K. (2010). Cellular and molecular dissection of pluripotent adult somatic stem cells in planarians. Development, Growth and Differentiation 52, 2741.
Skelly, P. J., Da'dara, A. and Harn, D. A. (2003). Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference. International Journal for Parasitology 33, 363369.
Sliva, K. and Schnierle, B. S. (2010). Selective gene silencing by viral delivery of short hairpin RNA. Virology Journal, 7, 248.
Spiliotis, M., Mizukami, C., Oku, Y., Kiss, F., Brehm, K. and Gottstein, B. (2010). Echinococcus multilocularis primary cells: improved isolation, small-scale cultivation and RNA interference. Molecular and Biochemical Parasitology 174, 8387.
Stefanic, S., Dvorak, J., Horn, M., Braschi, S., Sojka, D., Ruelas, D. S., Suzuki, B., Lim, K. C., Hopkins, S. D., McKerrow, J. H. and Caffrey, C. R. (2010). RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening. PLoS Neglected Tropical Diseases 4: e850.
Strebel, K., Luban, J. and Jeang, K. T. (2009). Human cellular restriction factors that target HIV-1 replication. BMC Medicine 7, 48.
Takeuchi, H. and Matano, T. (2008). Host factors involved in resistance to retroviral infection. Microbiology and Immunology 52, 318325.
Tchoubrieva, E. B., Ong, P. C., Pike, R. N., Brindley, P. J. and Kalinna, B. H. (2010). Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni. Cellular and Molecular Life Sciences 67, 37393748.
Turan, S., Galla, M., Ernst, E., Qiao, J., Voelkel, C., Schiedlmeier, B., Zehe, C. and Bode, J. (2011). Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. Journal of Molecular Biology 407, 193221.
van Ooij, C., Tamez, P., Bhattacharjee, S., Hiller, N. L., Harrison, T., Liolios, K., Kooij, T., Ramesar, J., Balu, B., Adams, J., Waters, A. P., Janse, C. J. and Haldar, K. (2008). The malaria secretome: from algorithms to essential function in blood stage infection. PLoS Pathogens 4: e1000084.
Wakiyama, M., Matsumoto, T. and Yokoyama, S. (2005). Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells. Biochemical and Biophysical Research Communications 331, 11631170.
Wilson, M. H., Coates, C. J. and George, A. L. Jr. (2007). PiggyBac transposon-mediated gene transfer in human cells. Molecular Therapy 15, 139145.
Wippersteg, V., Kapp, K., Kunz, W. and Grevelding, C. G. (2002 a). Characterisation of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae. International Journal for Parasitology 32, 12191224.
Wippersteg, V., Kapp, K., Kunz, W., Jackstadt, W. P., Zahner, H. and Grevelding, C. G. (2002 b). HSP70-controlled GFP expression in transiently transformed schistosomes. Molecular and Biochemical Parasitology 120, 141150.
Wippersteg, V., Ribeiro, F., Liedtke, S., Kusel, J. R. and Grevelding, C. G. (2003). The uptake of Texas Red-BSA in the excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males. International Journal for Parasitology 33, 11391143.
Wippersteg, V., Sajid, M., Walshe, D., Khiem, D., Salter, J. P., McKerrow, J. H., Grevelding, C. G. and Caffrey, C. R. (2005). Biolistic transformation of Schistosoma mansoni with 5′ flanking regions of two peptidase genes promotes tissue-specific expression. International Journal for Parasitology 35, 583589.
Wise, T. G., Schafer, D. J., Lambeth, L. S., Tyack, S. G., Bruce, M. P., Moore, R. J. and Doran, T. J. (2007). Characterization and comparison of chicken U6 promoters for the expression of short hairpin RNAs. Animal Biotechnology 18, 153162.
Yang, S., Brindley, P. J., Zeng, Q., Li, Y., Zhou, J., Liu, Y., Liu, B., Cai, L., Zeng, T., Wei, Q., Lan, L. and McManus, D. P. (2010). Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes. Molecular and Biochemical Parasitology 174, 109116.
Yuan, X. S., Shen, J. L., Wang, X. L., Wu, X. S., Liu, D. P., Dong, H. F. and Jiang, M. S. (2005). Schistosoma japonicum: a method for transformation by electroporation. Experimental Parasitology 111, 244249.
Yuan, Y. W. and Wessler, S. R. (2011). The catalytic domain of all eukaryotic cut-and-paste transposase superfamilies. Proceedings of the National Academy of Sciences, USA 108, 78847889.
Zhao, Z. R., Lei, L., Liu, M., Zhu, S. C., Ren, C. P., Wang, X. N. and Shen, J. J. (2008). Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference. Experimental Parasitology 119, 379384.

Keywords

Genetic manipulation of schistosomes – progress with integration competent vectors

  • SUTAS SUTTIPRAPA (a1), GABRIEL RINALDI (a1) (a2) and PAUL J. BRINDLEY (a1)

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