whole-mount in situ hybridization. zebrafish development

Whole-mount in situ Hybridization

Zebrafish Development

Function?

Over-expression knockdown

DNA: using tissue specific promoter

RNA: in vitro transcription

Morpholino

Dominant negative form

Antagonist

Whole mount in situ hybridization

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: C43H61N4O21P3Li4 Molecular Weight: 1090.7DIG-UTP (R1=OH, R2=OH) DIG-dUTP (R1=OH, R2=H) DIG-ddUTP (R1= H, R2=H)

Non-Radioactive RNA-Labeling

TA vector

SP6 T7Probe

HindIII EcoRIHindIII

Detection methods

• Chemiluminescent detection

CDP-star or CSPD

• Colorimetric detection

NBT/BCIP

• Fluorescent detection

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

• http://zfin.org

• 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: 139-149)

(Nature Cell Biology 2002, 4: 170-174)

(Mechanisms of Development 2002, 119: 91-108)

(Development 2002, 129: 917-933)