Fast Release Clones: A High Throughput Expression Analysis

Method:

• Embryos from AB/TU fish (a strain generated from crosses of two wild-type lines, AB and TU) are collected, dechorionated by pronase treatment, allowed to develop at 28.5°C until the appropriate stage and then fixed by incubation over night in 4% paraformaldehyde at 4°C. Embryos older than 24h (hours post fertilization) are incubated in 0.3x Danieau medium supplemented with 1-phenyl-2-thiourea (PTU, 0.003%) to prevent accumulation of pigment. After fixation, embryos are dehydrated and stored at -20°C in 100% methanol prior to in situ hybridization.
  The in situ hybridization is performed according to Thisse, C. and Thisse, B. (1998). High resolution whole-mount in situ hybridization. Zebrafish Science Monitor, vol 5. Eugene: University of Oregon Press.
  The labeling reaction is monitored under a dissecting microscope and the reaction is stopped with 1x PBS at pH 5.5. Embryos are then mounted in 100% glycerol and incubated at least 24 hours in the dark at room temperature prior to observation. Embryos are mounted under a coverslip in 100% glycerol. Pictures are taken using a color CCD camera (Roper Scientific, Coolsnap) mounted on a dissecting microscope (Leica, M420) or on a compound microscope (Leica, DM RA2HC or Nikon, FXA).
• cDNAs obtained from various zebrafish cDNA libraries are used as templates for the preparation of digoxygenin labeled antisense RNA probes. Plasmid DNA is purified and PCR amplification carried out for 35 cycles using the PCR primers listed on the individual probe details page. PCR reaction mixture is denatured for 4 min. followed by PCR cycling 95°C 30s, 55°C 30s, 72°C 3 min. (at least 1 min. per kb), followed by elongation at 72°C for 7 min. PCR reactions are purified and used to synthesize antisense RNA.
• cDNA libraries used:
  • 7 embryonic stages pooled: SMART Sfi-(dT)30 primed library, GISZF001. Constructed by S. Mathavan, Chia-Lin Wei and Yijun Ruan (Genome Institute of Singapore)
  • ZGC clones: Full length cDNA library from the ZGC project at NIH.
  • I.M.A.G.E. clones: zebrafish ESTs from the I.M.A.G.E. consortium.

Acknowledging this work:
All clones, information, and images used from this work should cite this summary. If you have any questions or comments on this project, please contact C and B Thisse.