Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2001 Mar;157(3):1217–1226. doi: 10.1093/genetics/157.3.1217

Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.

V Praitis 1, E Casey 1, D Collar 1, J Austin 1
PMCID: PMC1461581  PMID: 11238406

Abstract

In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing.

Full Text

The Full Text of this article is available as a PDF (243.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armaleo D., Ye G. N., Klein T. M., Shark K. B., Sanford J. C., Johnston S. A. Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi. Curr Genet. 1990 Feb;17(2):97–103. doi: 10.1007/BF00312852. [DOI] [PubMed] [Google Scholar]
  2. Broverman S., MacMorris M., Blumenthal T. Alteration of Caenorhabditis elegans gene expression by targeted transformation. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4359–4363. doi: 10.1073/pnas.90.10.4359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cassada R. C., Russell R. L. The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev Biol. 1975 Oct;46(2):326–342. doi: 10.1016/0012-1606(75)90109-8. [DOI] [PubMed] [Google Scholar]
  4. Cassidy-Hanley D., Bowen J., Lee J. H., Cole E., VerPlank L. A., Gaertig J., Gorovsky M. A., Bruns P. J. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics. 1997 May;146(1):135–147. doi: 10.1093/genetics/146.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christou P., McCabe D. E., Swain W. F. Stable Transformation of Soybean Callus by DNA-Coated Gold Particles. Plant Physiol. 1988 Jul;87(3):671–674. doi: 10.1104/pp.87.3.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fire A. Integrative transformation of Caenorhabditis elegans. EMBO J. 1986 Oct;5(10):2673–2680. doi: 10.1002/j.1460-2075.1986.tb04550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fire A., Waterston R. H. Proper expression of myosin genes in transgenic nematodes. EMBO J. 1989 Nov;8(11):3419–3428. doi: 10.1002/j.1460-2075.1989.tb08506.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hsieh J., Liu J., Kostas S. A., Chang C., Sternberg P. W., Fire A. 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. 1999 Nov 15;13(22):2958–2970. doi: 10.1101/gad.13.22.2958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jackstadt P., Wilm T. P., Zahner H., Hobom G. Transformation of nematodes via ballistic DNA transfer. Mol Biochem Parasitol. 1999 Oct 15;103(2):261–266. doi: 10.1016/s0166-6851(99)00089-4. [DOI] [PubMed] [Google Scholar]
  10. Kelly W. G., Fire A. Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans. Development. 1998 Jul;125(13):2451–2456. doi: 10.1242/dev.125.13.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kelly W. G., Xu S., Montgomery M. K., Fire A. Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics. 1997 May;146(1):227–238. doi: 10.1093/genetics/146.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kindle K. L., Schnell R. A., Fernández E., Lefebvre P. A. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol. 1989 Dec;109(6 Pt 1):2589–2601. doi: 10.1083/jcb.109.6.2589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krause M., Harrison S. W., Xu S. Q., Chen L., Fire A. Elements regulating cell- and stage-specific expression of the C. elegans MyoD family homolog hlh-1. Dev Biol. 1994 Nov;166(1):133–148. doi: 10.1006/dbio.1994.1302. [DOI] [PubMed] [Google Scholar]
  14. MacMorris M., Spieth J., Madej C., Lea K., Blumenthal T. Analysis of the VPE sequences in the Caenorhabditis elegans vit-2 promoter with extrachromosomal tandem array-containing transgenic strains. Mol Cell Biol. 1994 Jan;14(1):484–491. doi: 10.1128/mcb.14.1.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maduro M., Pilgrim D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics. 1995 Nov;141(3):977–988. doi: 10.1093/genetics/141.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McKim K. S., Howell A. M., Rose A. M. The effects of translocations on recombination frequency in Caenorhabditis elegans. Genetics. 1988 Dec;120(4):987–1001. doi: 10.1093/genetics/120.4.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Okkema P. G., Harrison S. W., Plunger V., Aryana A., Fire A. Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics. 1993 Oct;135(2):385–404. doi: 10.1093/genetics/135.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rosenbluth R. E., Baillie D. L. The genetic analysis of a reciprocal translocation, eT1(III; V), in Caenorhabditis elegans. Genetics. 1981 Nov-Dec;99(3-4):415–428. doi: 10.1093/genetics/99.3-4.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rosenbluth R. E., Johnsen R. C., Baillie D. L. Pairing for recombination in LGV of Caenorhabditis elegans: a model based on recombination in deficiency heterozygotes. Genetics. 1990 Mar;124(3):615–625. doi: 10.1093/genetics/124.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seydoux G., Strome S. Launching the germline in Caenorhabditis elegans: regulation of gene expression in early germ cells. Development. 1999 Aug;126(15):3275–3283. doi: 10.1242/dev.126.15.3275. [DOI] [PubMed] [Google Scholar]
  21. Spieth J., MacMorris M., Broverman S., Greenspoon S., Blumenthal T. Regulated expression of a vitellogenin fusion gene in transgenic nematodes. Dev Biol. 1988 Nov;130(1):285–293. doi: 10.1016/0012-1606(88)90434-4. [DOI] [PubMed] [Google Scholar]
  22. Stinchcomb D. T., Shaw J. E., Carr S. H., Hirsh D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol Cell Biol. 1985 Dec;5(12):3484–3496. doi: 10.1128/mcb.5.12.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thatcher J. D., Haun C., Okkema P. G. The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx. Development. 1999 Jan;126(1):97–107. doi: 10.1242/dev.126.1.97. [DOI] [PubMed] [Google Scholar]
  24. Williams B. D., Schrank B., Huynh C., Shownkeen R., Waterston R. H. A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Genetics. 1992 Jul;131(3):609–624. doi: 10.1093/genetics/131.3.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wilm T., Demel P., Koop H. U., Schnabel H., Schnabel R. Ballistic transformation of Caenorhabditis elegans. Gene. 1999 Mar 18;229(1-2):31–35. doi: 10.1016/s0378-1119(99)00043-8. [DOI] [PubMed] [Google Scholar]
  26. Zelenin A. V., Alimov A. A., Titomirov A. V., Kazansky A. V., Gorodetsky S. I., Kolesnikov V. A. High-velocity mechanical DNA transfer of the chloramphenicolacetyl transferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo. FEBS Lett. 1991 Mar 11;280(1):94–96. doi: 10.1016/0014-5793(91)80212-l. [DOI] [PubMed] [Google Scholar]
  27. Zetka M. C., Rose A. M. The meiotic behavior of an inversion in Caenorhabditis elegans. Genetics. 1992 Jun;131(2):321–332. doi: 10.1093/genetics/131.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES