whole-mount in situ hybridization. zebrafish development

17
Whole-mount in situ Hybridization

Post on 19-Dec-2015

242 views

Category:

Documents


4 download

TRANSCRIPT

Page 1: Whole-mount in situ Hybridization. Zebrafish Development

Whole-mount in situ Hybridization

Page 2: Whole-mount in situ Hybridization. Zebrafish Development

Zebrafish Development

Page 3: 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

Page 4: Whole-mount in situ Hybridization. Zebrafish Development

Whole mount in situ hybridization

Page 5: Whole-mount in situ Hybridization. Zebrafish Development

Digoxigenin (DIG)

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

Page 6: Whole-mount in situ Hybridization. Zebrafish Development

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)

Page 7: Whole-mount in situ Hybridization. Zebrafish Development

Non-Radioactive RNA-Labeling

Page 8: Whole-mount in situ Hybridization. Zebrafish Development
Page 9: Whole-mount in situ Hybridization. Zebrafish Development

TA vector

SP6 T7Probe

HindIII EcoRIHindIII

Page 10: Whole-mount in situ Hybridization. Zebrafish Development

Detection methods

• Chemiluminescent detection

CDP-star or CSPD

• Colorimetric detection

NBT/BCIP

• Fluorescent detection

Page 11: Whole-mount in situ Hybridization. Zebrafish Development

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

• http://zfin.org

Page 12: Whole-mount in situ Hybridization. Zebrafish Development

• mRNA: FGF8 (Fibroblast Growth Factor 8)

• Probe: Sef (Similar Expression of Fgf gene)

Page 13: Whole-mount in situ Hybridization. Zebrafish Development

(Endocrine Reviews 2005, 26: 63-77)

Page 14: Whole-mount in situ Hybridization. Zebrafish Development

(Cytokine & Growth Factor Reviews 2005, 16: 139-149)

Page 15: Whole-mount in situ Hybridization. Zebrafish Development

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

Page 16: Whole-mount in situ Hybridization. Zebrafish Development

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

Page 17: Whole-mount in situ Hybridization. Zebrafish Development

(Development 2002, 129: 917-933)