Abstract
Introduction of exogenous DNA into Caenorhabditis elegans is important for examining the expression of altered or reporter gene constructs, rescuing mutant genes, and studying gene function in vivo. Until recently, germ-line injection was the most commonly used method for transforming C. elegans strains. This chapter describes four different microparticle bombardment methods used to transform C. elegans with exogenous DNA. We include a discussion of the advantages and disadvantages of using micro-particle bombardment for transformation, list cotransformation markers that have been used successfully in microparticle bombardment experiments, and discuss transformation efficiency.
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Mello, C. and Fire, A. (1995) DNA transformation. Methods Cell Biol. 48, 451–482.
Stinchcomb, D. T., Shaw, J. E., Carr, S. H., and Hirsh, D. (1985) Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484–3496.
Fire, A. and Waterston, R. H. (1989) Proper expression of myosin genes in transgenic nematodes. EMBO J. 8, 3419–3428.
Mello, C. C., Kramer, J. M., Stinchcomb, D., and Ambros. V. (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970.
MacMorris, M., Spieth, J., Madej, C., Lea, K., and Blumenthal, T. (1994) Analysis of the VPE Sequences in the Caenorhabditis elegans vit-2 promotor with extrachromosomal tandem array-containing transgenic strains. Mol. Cell. Biol. 14, 484–491.
Hsieh, J., Liu, J., Kostas, S. A., Chang, C., Sternberg, P. W., and Fire, A. (1999) The Ring finger/B-Box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans. Genes Dev. 13, 2958–2970.
Kelly, W. G., Xu, S., Montgomery, M. K., and Fire, A. (1997) Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene. Genetics 146, 227–238.
Krause, M., Harrison, S. W., Xu, S. Q., Chen, L., and Fire, A. (1994) Elements regulating cell and stage-specific expression of the C. elegans MyoD family homolog hlh-1. Dev. Biol. 166, 133–148.
Fire, A. (1986) Integrative transformation of Caenorhabditis elegans. EMBO J. 5, 2673–2680.
Spieth, J., MacMorris, M., Broverman, S., Greenspoon, S., and Blumenthal, T. (1988) Regulated expression of a vitellogenin fusion gene in transgenic nematodes. Dev. Biol. 130, 285–293.
Barrett, P. L., Fleming, J. T., and Gobel, V. (2004) Targeted gene alteration in Caenorhabditis elegans by gene conversion. Nat. Genet. 36, 1231–1237.
Strome, S., Powers, J., Dunn, M., et al. (2001) Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos. Mol. Biol. Cell 12, 1751–1764.
Jackstadt, P., Wilm, T. P., Zahner, H., and Hobom, G. (1999) Transformation of nematodes via ballistic DNA transfer. Mol. Biochem. Parasitol. 103, 261–266.
Wilm, T., Demel, P., Koop, H. U., Schnabel, H., and Schnabel, R. (1999) Ballistic transformation of Caenorhabditis elegans. Gene 229, 31–35.
Praitis, V., Casey, E., Collar, D., and Austin, J. (2001) Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226.
Berezikov, E., Bargmann, C., and Plasterk, R. (2004) Homologous gene targeting in Caenorhabditis elegans by biolistic transformation. Nucleic Acids Res. 32
Johnson, N. M., Behm, C. A., and Trowell, S. C. (2005) Heritable and inducible gene knockdown in C. elegans using Wormgate and the ORFeome. Gene 10, 26–34.
Davis, R. E., Parra, A., Lo Verde, P. T., Ribeiro, E., Glorioso, G., and Hodgson, S. (1999) Transient expression of DNA and RNA in parasitic helminths by using particle bombardment. Proc. Natl. Acad. Sci. USA 96, 8687–8692.
Jackstadt, P., Zahner, H., and Hobom, G. (1999) Bolistic particle delivery. Transformation nematodes with the Helios™ gene gun. Bio-Rad Technical Note 2433.
Askjaer, P., Galy, V., Hannak, E., and Mattaj. I. (2002) Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. Mol. Biol. Cell 13, 4355–4570.
Trappe, R., Schulze, E., Rzymski, T., Frode, S., and Engel, W. (2002) The Caenorhabditis elegans ortholog of human PHF5a shows a muscle-specific expression domain and is essential for C. elegans morphogenetic development. Biochem. Biophys. Res. Commun. 297, 1049–1057.
Kirkham, M., Muller-Reichert, T., Oegema, K., Grill, S., and Hyman, A. (2003) SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112, 575–587.
Pellettieri, J., Reinke, V., Kim, S., and Seydoux, G. (2003) Coordinate activation of maternal protein degradation during the egg-to-embryo transition in C. elegans. Dev. Cell 5, 451–462.
Grad, L., and Lemire, B. (2004) Mitochondrial complex I mutations in Caenorhabditis elegans produce cytochrome c oxidase deficiency, oxidative stress and vitamin-responsive lactic acidosis. Hum. Mol. Gen. 13, 303–314.
Maduro, M. and Pilgrim, D. (1995) Identification and Cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics 141, 977–988.
Granato, M., Schnabel H., and Schnabel, R. (1994) pha-1, a selectable marker for gene-transfer in C. elegans. Nucleic Acids Res. 22, 1762–1763.
Varkey, J., Muhlrad, P., Minniti, A., Do B.K., and Ward, S. (1995) The Caenorhabditis elegans spe-26 gene is necessary to form spermatids and encodes a protein similar to the actin-associated proteins Kelch and Scruin. Genes Dev. 9, 1074–1086.
Clark, D. V., Suleman, D. S., Beckenbach, K. A., Gilchrist E. J., and Baillie, D. L. (1995) Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans. Mol. Gen. Genet. 247, 367–378.
Kniss, S. (2002). The role of the sue genes in the neuronal regulation of C. elegans behavior. PhD Thesis, University of Chicago, Chicago, IL.
Kramer, J. M., French, R. P., Park, E.C., and Johnson, J. J. (1990) The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collage gene to determine organismal morphology, encodes a collagen. Mol. Cell Biol. 10, 2081–2089.
Zwaal, R. R., Van Baelen, K., Groenen, J. T., et al. (2001) The sarco-endoplasmic reticulum Ca2+ ATPase is required for development and muscle function in Caenorhabditis elegans. J. Biol. Chem. 276, 43,557–43,563.
Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71–94.
Lewis, J. A. and Flemming, J. T. (1995) Basic culture methods, in Caenorhabditis elegans: Modern Biological Analysis of an Organism, (H. F. Epstein and D. C. Shakes, eds.), Academic Press, San Diego, CA, pp. 3–29.
Bio-Rad (1997) Bio-Rad Biolistic® PDS-1000/He Particle Delivery System Instruction Manual, Hercules, CA.
Bio-Rad. Bio-Rad Hepta Adapter Instruction Manual, Hercules, CA.
Finer, J. J., Vain, P., Johns M. W., and McMullen, M. D. (1992) Development of the particle gun for the delivery to plant cells. Plant Cell Rep. 11, 323–328.
Takeuchi, Y., Dotson, M., and Keen, N. T. (1992) Plant transformation: a simple particle bombardment device based on flowing helium. Plant Mol. Biol. 18, 835–839.
Bio-Rad (2001) Bio-Rad Helios Gene Gun System Instruction Manual, Hercules, CA.
Sambrook, J., Fritsch, E. F., and Maniatis. T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Praitis, V. (2006). Creation of Transgenic Lines Using Microparticle Bombardment Methods. In: Strange, K. (eds) C. elegans. Methods in Molecular Biology, vol 351. Humana Press. https://doi.org/10.1385/1-59745-151-7:93
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DOI: https://doi.org/10.1385/1-59745-151-7:93
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