Whole-mount in situ Hybridization. Zebrafish Development.

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Presentation transcript:

Whole-mount in situ Hybridization

Zebrafish Development

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

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

Structure of DIG-Modified Nucleotides Coupled via Alkali-Stable Ether Bond Formula: C 43 H 61 N 4 O 21 P 3 Li 4 Molecular Weight: DIG-UTP (R1=OH, R2=OH) DIG-dUTP (R1=OH, R2=H) DIG-ddUTP (R1= H, R2=H)

Non-Radioactive RNA-Labeling

TA vector SP6T7 Probe HindIII EcoRI HindIII

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

science.com/prodinfo_fst.htm?/DIG/dig_la beling03.htm

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

(Endocrine Reviews 2005, 26: 63-77)

(Cytokine & Growth Factor Reviews 2005, 16: )

(Nature Cell Biology 2002, 4: )

(Mechanisms of Development 2002, 119: )

(Development 2002, 129: )