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Biology

Een snelle protocol voor de integratie Extrachromosomaal Arrays Met High Transmission Rate in de Published: December 9, 2013 doi: 10.3791/50773

Materials

Name Company Catalog Number Comments
Agar Prolabo 20768.292 for NGM (nematode growth medium) preparation (6,7)
NaCl Chimie-Plus 40045 for NGM preparation
Bacto-peptone BD 211820 for NGM preparation
KH2PO4 Roth 3904-1 for NGM preparation
K2HPO4 Prolabo 26930.293 for NGM preparation
MgSO4 Roth P027.2 for NGM preparation
CaCl2 Prolabo 22317.297 for NGM preparation
Cholesterol Rectapur for NGM preparation
LB-broth Sigma L3022 for culture of OP50 bacteria
EQUIPMENT
U.V. cross-linker Fischer Scientific Wavelength 254 nm
Microscope SteREO Discovery V8 Carl Zeiss, Inc.
Petri dishes, 60 mm CML BPS55E6
Platinium wire 0.25 mm Alfa Aesar 7440-06-4 To make a pick for worms (7)

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References

  1. Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. Generation of stable transgenic C. elegans using microinjection. J. Vis. Exp. (18), e833 (2008).
  2. Mello, C., Kramer, J. M., Stinchlomb, D., Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10 (12), 3959-3970 (1991).
  3. Transformation and microinjection. WormBook. Evans, T. C. , The C. elegans Research Community, WormBook. (2006).
  4. Hobert, O. PCR Fusion-Based Approach to Create Reporter Gene Constructs for Expression Analysis in Transgenic C. elegans. BioTechniques. 32, 728-730 (2002).
  5. Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484-3496 (1985).
  6. Brenner, S. The genetics of Caenorhabditis elegans. Genetics. 77, 71-94 (1974).
  7. Chaudhuri, J., Parihar, M., Pires-daSilva, A. An Introduction to Worm Lab: from Culturing Worms to Mutagenesis. J. Vis. Exp. (47), e2293 (2011).
  8. Merritt, C., Rasoloson, D., Gallo, C., Voronina, E., Ko, D., Seydoux, G. A modular vector kit for transgenesis in C. elegans. International Worm Meeting. , (2007).
  9. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Green Prasher, D. Fluorescent protein as a marker for gene expression. Science. 263, 802-805 (1994).
  10. Fleenor, J., Fire, A. Discosoma sp. Red Fluorescent Protein (dsRED) as a reporter in C. elegans. Worm Breeder's Gazette. 16 (3), 16 (2000).
  11. Lecroisey, C., et al. ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration. Mol. Biol. Cell. , (2013).
  12. Praitis, V., Casey, E., Collar, D., Austin, J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics. 157 (3), 1217-1226 (2001).
  13. Frøkjaer-Jensen, C., et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40 (11), 1375-1383 (2008).
  14. Dickinson, D. J., Ward, J. D., Reiner, D. J., Goldstein, B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods. 10 (10), 1028-1034 (2013).
Een snelle protocol voor de integratie Extrachromosomaal Arrays Met High Transmission Rate in de<em&gt; C. elegans</em&gt; Genome
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Cite this Article

Mariol, M. C., Walter, L., Bellemin, More

Mariol, M. C., Walter, L., Bellemin, S., Gieseler, K. A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome. J. Vis. Exp. (82), e50773, doi:10.3791/50773 (2013).

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