Whole-mount in situ Hybridization
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Transcript of Whole-mount in situ Hybridization
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Whole-mount in situ Hybridization
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Zebrafish Development
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Function?
Over-expression knockdown
DNA: using tissue specific promoter
RNA: in vitro transcription
Morpholino
Dominant negative form
Antagonist
Whole mount in situ hybridization
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Whole mount in situ hybridization
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Digoxigenin (DIG)
• DIG is a steroid hapten from Digitalis lanata (毛地黃 )
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Structure of DIG-Modified Nucleotides Coupled via Alkali-Stable Ether Bond
Formula: C43H61N4O21P3Li4 Molecular Weight: 1090.7DIG-UTP (R1=OH, R2=OH) DIG-dUTP (R1=OH, R2=H) DIG-ddUTP (R1= H, R2=H)
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Non-Radioactive RNA-Labeling
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TA vector
SP6 T7Probe
HindIII EcoRIHindIII
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Detection methods
• Chemiluminescent detection
CDP-star or CSPD
• Colorimetric detection
NBT/BCIP
• Fluorescent detection
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• http://www.roche-applied-science.com/prodinfo_fst.htm?/DIG/dig_labeling03.htm
• http://zfin.org
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• mRNA: FGF8 (Fibroblast Growth Factor 8)
• Probe: Sef (Similar Expression of Fgf gene)
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(Endocrine Reviews 2005, 26: 63-77)
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(Cytokine & Growth Factor Reviews 2005, 16: 139-149)
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(Nature Cell Biology 2002, 4: 170-174)
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(Mechanisms of Development 2002, 119: 91-108)
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(Development 2002, 129: 917-933)