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Whole-mount in situ Hybridization

Whole-mount in situ Hybridization. Zebrafish Development. Function?. Over-expression. knockdown. DNA: using tissue specific promoter. Morpholino. RNA: in vitro transcription. Dominant negative form. Antagonist. Whole mount in situ hybridization. Whole mount in situ hybridization.

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Whole-mount in situ Hybridization

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  1. Whole-mount in situ Hybridization

  2. Zebrafish Development

  3. Function? Over-expression knockdown DNA: using tissue specific promoter Morpholino RNA: in vitro transcription Dominant negative form Antagonist Whole mount in situ hybridization

  4. Whole mount in situ hybridization

  5. Digoxigenin (DIG) • DIG is a steroid hapten from Digitalis lanata (毛地黃)

  6. Structure of DIG-Modified Nucleotides Coupled via Alkali-Stable Ether Bond Formula: C43H61N4O21P3Li4 Molecular Weight: 1090.7 DIG-UTP (R1=OH, R2=OH) DIG-dUTP (R1=OH, R2=H) DIG-ddUTP (R1= H, R2=H)

  7. Non-Radioactive RNA-Labeling

  8. HindIII HindIII EcoRI SP6 T7 Probe TA vector

  9. Detection methods • Chemiluminescent detection  CDP-star or CSPD • Colorimetric detection  NBT/BCIP • Fluorescent detection

  10. http://www.roche-applied-science.com/prodinfo_fst.htm?/DIG/dig_labeling03.htmhttp://www.roche-applied-science.com/prodinfo_fst.htm?/DIG/dig_labeling03.htm • http://zfin.org

  11. mRNA: FGF8 (Fibroblast Growth Factor 8) • Probe: Sef (Similar Expression of Fgf gene)

  12. (Endocrine Reviews2005, 26: 63-77)

  13. (Cytokine & Growth Factor Reviews2005, 16: 139-149)

  14. (Nature Cell Biology2002, 4: 170-174)

  15. (Mechanisms of Development2002, 119: 91-108)

  16. (Development2002, 129: 917-933)

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