Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
권호 표지

Isolation of Deletion Mutants by Reverse Genetics in Caenorhabditis elegans

  • Park, Byung-Jae (Department of Life Science, Kwangju Institute of Science and Technology) ;
  • Lee, Jin ll (Department of Life Science, Kwangju Institute of Science and Technology) ;
  • Lee, Jiyeon (Department of Life Science, Kwangju Institute of Science and Technology) ;
  • Kim, Sunja (Department of Life Science, Kwangju Institute of Science and Technology) ;
  • Choi, Kyu Yeong (Department of Life Science, Kwangju Institute of Science and Technology) ;
  • Park, Chul-Seung (Department of Life Science, Kwangju Institute of Science and Technology) ;
  • Ahn, Joohong (Department of Life Science, Kwangju Institute of Science and Technology)
  • Published : 2001.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Obtaining mutant animals is important for studying the function of a particular gene. A chemical mutagenesis was first carried out to generate mutations in C. elegans. In this study, we used ultraviolet-activated 4,5',8-trimethylpsoralen to induce small deletion mutations. A library of mutagenized worms was prepared for recovery of candidate animals and stored at $15^{\circ}C$ during screening instead of being made into a frozen stock library. In order to isolate deletion mutations in target genes, a polymerase chain reaction (PCR)-based screening method was used. As a result, two independent mutants with deletions of approximately 1.0 kb and 1.3 kb were isolated. This modified and improved reverse genetic approach was proven to be effective and practical for isolating mutant animals to study gene function at the organismal level.

Keywords

References

  1. Anderson P (1995) Mutagenesis. In: Epstein HF and Shakes DC (eds), Caenorhabditis elegans: Modern Biological Analysis of an Organism, Methods Cell Biol 48: 31-58
  2. Barstead RJ (1999) Reverse Genetics. In: Hope IA (ed), C. elegans: a Practical Approach, Oxford University Press, New York, pp 97-118
  3. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71-94
  4. Cho JH, Oh YS, Park KW, Yu JR, Choi KY, Shin JY, Kim DH, Park WJ, Hamada T, Kagawa H, Maryon EB, Bandyopadhyay J, and Ahnn J (2000) Calsequestrin, a calcium sequestering protein localized at the sarcoplasmic reticulum is not essential for body-wall muscle formation in Caenorhabditis elegans. J Cell Sci 113: 3947-3958
  5. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, and Mello C (1988) Potent and specific genetic interference by double-stranded RNA in Caneorhabditis elegans. Nature 391: 801-811 https://doi.org/10.1038/35888
  6. Genyo-Ando K and Mitani S (2000) Characterization of mutations induced by ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans. Biochem Biophys Res Commun 269: 64-69 https://doi.org/10.1006/bbrc.2000.2260
  7. Jansen G, Hazendonk E, Thijssen KL, and Plasterk RH (1997) Reverse genetics by chemical mutagenesis in Caenorhabditis elegans. Nat Genet 17: 119-121 https://doi.org/10.1038/ng0997-119
  8. Liu L, Spoerke JM, Mulligan EL, Chen J, Reardon B, Westlund B, Sun L, Abel K, Armstrong B, Hardiman G, King J, McCague L, Basson M, Clover R, and Johnson CD (1999) High-throughput isolation of Caenhorabditis elegans deletion mutants. Genome Res 9: 859-867 https://doi.org/10.1101/gr.9.9.859
  9. Rosenbluth R, Cuddeford C, and Bailie D (1983) Mutagenesis in C. elegans. I. A rapid eukaryotic mutagen test system using the reciprocal translocation eT1 (IIIV). Mutat Res 110: 39-48 https://doi.org/10.1016/0027-5107(83)90016-7
  10. Sulston J and Hodgkin J (1988) Methods. In: Wood WB (ed), The Nematode Caenorhabditis elegans, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 281-335
  11. The C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282: 2012-2018 https://doi.org/10.1126/science.282.5396.2012
  12. Yandell MD, Edgar LG, and Wood WB (1994) Trimethylpsoralen induces small deletion mutations in Caenorhabditis elegans. Proc Natl Acad Sci USA 91: 1381-1385 https://doi.org/10.1073/pnas.91.4.1381
  13. Zwaal RR, Broeks A, van Meurs J, Groenen JTM, and Plasterk RHA (1993) Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank. Proc Nat Aca Sci USA 90: 7431-7435 https://doi.org/10.1073/pnas.90.16.7431