Functional genomics a new addition to chinese medicine research development
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Functional genomics — a new addition to Chinese Medicine Research & Development. (CM2006, November 24, Hong Kong). Yuhui Hu Max-Planck-Institute for Molecular Genetics Berlin, Germany Dept. Vertebrate Genomics (Prof. Hans Lehrach ). Accomplishment in human genome sequencing

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Functional genomics — a new addition to Chinese Medicine Research & Development

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Functional genomics a new addition to chinese medicine research development

Functional genomics —a new addition to Chinese Medicine Research & Development

(CM2006, November 24, Hong Kong)

Yuhui Hu

Max-Planck-Institute for Molecular Genetics

Berlin, Germany

Dept. Vertebrate Genomics (Prof. Hans Lehrach)


Functional genomics a new addition to chinese medicine research development

Accomplishment in human genome sequencing

~ 3 billion nucleotides

~ contain 20–25,000 protein-coding genes

Nature (2001)

Science (2001)

Nature (2004)


Functional genomics a new addition to chinese medicine research development

New focus in post-genomic era:

Functional investigation of DNA sequences

Genome

Phenotype

Environment


Functional genomics a new addition to chinese medicine research development

Genome

……

Phenotype

Environment

DNA

Transcript

(mRNA)

Protein

Functional Genomics any approach that converts the information encoded in DNA sequences into an understanding of gene functions and effects.


Functional genomics a new addition to chinese medicine research development

Nano-well PCR & qPCR (TaqMan) 200nl reaction (Group Nyarsik)

Gene expression patterns—mouse embryo

High-throughput in situ hybridization~17,000 clones & probes

(EUREXPRESS consortium) (Group Yaspo)

Our functional genomic tools

Genomics:DNA sequence; SNP; Duplications

1st , 2nd generation of DNA sequencers; DNA microarray

Epigenomics:DNA methylation; Histone code and it’s impact on gene expression

Chromatin immunoprecipitation + Genomic arrays (ChIP-Chip)

Transcriptome:

Gene expression profiling

Proteomics:

Expression— High-throughput cloning & expression strategy

Structure of protein complex— LC-MALDI MS (Group Gobom)

Cryo-electron microscopy (Group Mielke)

Function— Protein localization; Protein-protein interaction

Transfected cell microarray (Group Janitz)

Mammalian & Yeast two hybridization

in vitro ligand screening; Protein & Antibody array (Group Konthur)

Protein expression & structure (Group Büssow)


Functional genomics a new addition to chinese medicine research development

Regulation networks:

Genome-wide promoter analysis—

High-throughput evaluation of promoter activity

Transcription regulatory networks—

Knockdown all human Transcription Factors (TF)

Expression profiling

Genome location of TF binding sites (ChIP-on-Chip)

Bioinformatics:

Systems biology modelling

Signal transduction networks


Functional genomics a new addition to chinese medicine research development

……

Chinese medicine

Genome

Phenotype

Environment

DNA

Transcript

Protein

Genetics of human diseases (neurodegenerative & cardiovascular diseases; stroke; trisomy 21; etc)

Functional genomics tools


Functional genomics a new addition to chinese medicine research development

……

Genome

Phenotype

Environment

DNA

Transcript

Protein

Therapeutic mechanism

Chinese medicine

Diseases

Functional genomics tools


Functional genomics a new addition to chinese medicine research development

New drugs

  • Organism level

  • (Mouse, Rat, etc.)

Screening

at

Effective compounds

  • Cell level

DNA

Transcript

Protein

(Transfected Cell Microarray)

Therapeutic mechanism

Chinese medicine

Diseases

Functional genomics tools


Functional genomics a new addition to chinese medicine research development

Principle of Cell Microarray

pEXP26-C21orf

Manual or robot Spotting

Reverse transfection

Hek293 / HeLa

Expression vectors

siRNA or

shRNA vectors

High-throughput Functional Detections

Ziauddin and Sabatini, Nature, 2001

Hu, Y.H., et al., Biochemical Society Transaction,2005


Functional genomics a new addition to chinese medicine research development

Apoptosis

100x fluorescence

Localization

BioCCD scanning green/red

1000x Confocal / fluorescence

Applications of Cell Microarray

Labelled cell array

25x fluorescence

Hu, Y.H., et al., BMC Genomics, 2006


Functional genomics a new addition to chinese medicine research development

Protein co-localization with 9 organelles

Localization Categories

(MX1) Cytosol NOT co-ER

(HLCS) Cytosol

(PCBP3)Nucleus+Cytosol

(TMPRSS3) ER, co-ER

(HMGN1)Nucleoplasm

(NNP1)Nucleolus

(KCNJ6)PM

(KCNE2)Lysosome

The localization patterns provide the first hints to the functions.

Red: Chr21ORF Blue: Nucleus (DAPI) Green: organelle stain


Functional genomics a new addition to chinese medicine research development

Intracellular trafficking on cell array

6xHis-Chr21 Compartments Merged

gene: KCNE2

1. Golgi vesicles

Golgi

2.Plasma membrane

Mito

3. Plasma membrane & Lysosome/Endosome

Lysosome


Functional genomics a new addition to chinese medicine research development

Nucleus (M phase)

Nuc

Cell cycle-related translocationon cell array

6xHis-Chr21 Compartments Merged

gene: BACH1

Cytoplasm (interphase)

Nuc

BACH1: BTB and CNC homology 1 (transcription factor 1)


Functional genomics a new addition to chinese medicine research development

Cell deathdetection

Green:CLDN-14Red: Annexin V

Red:CLDN-14Green: TUNEL

Alteration of cell morphology due to protein overexpression

CLDN-14 ER + Nuclei Merged

gene: CLDN-14

Protein localization: ER

Cell retracted; Nuclear shape altered

CLDN14: Claudin-14 (Component of tight junction (TJ) strands )


Functional genomics a new addition to chinese medicine research development

siLaminA/C

Control siRNA

Bax over-expression

siCyclinB1

siSGTA

EGFP frame

Apoptosis due to gene overexpression & knockdown

Apoptosis staining:

Annexin V-Alexa488

TUNEL-Alexa488

Cleaved-Caspase3 Ab-Alexa488

SGTA: small glutamine-rich tetratricopeptide repeat-containing, alpha


Functional genomics a new addition to chinese medicine research development

Subcellular

localization

siRNA-mediated gene silencing

Cell phenotypes, e.g. apoptosis, proliferation

Applications of Cell Microarray

Cell Microrray

High-throughput

Functional Evaluation of

cDNA, sh/siRNA

High-throughput

Evaluation of

promotor activity

Large-scale cDNA/siRNA/shRNA libraries

Predicted promotor regions

Protein-protein

interactions

Cell type-specific

gene expression


Functional genomics a new addition to chinese medicine research development

Transfected Cell Microarray

  • robust

  • high-throughput expression & knockdown & detection

For Chinese Medicine

  • Therapeutic compound screening:

  • Cell death inducer — potential anti-cancer drug

  • Inhibitor of disease-related protein translocation & interaction,

  • e.g. transcription factors (NF-B), cell surface receptors (e.g. -Amyloid)

  • Multiple targets of Chinese Medicine—complexity and holism


Functional genomics a new addition to chinese medicine research development

New drugs

Screening

  • Organ level

Effective compounds

(Organotypic slice culture)

  • Organism level

  • (Mouse, Rat, etc.)

  • in vivo, low-throughput

at

  • Cell level

    (Cell microarray)

    in vitro, high-throughput

Chinese medicine


Functional genomics a new addition to chinese medicine research development

Organotypic Brain Slice Culture — Hippocampus

Brain

Dissection

P0-P9

hippocampus

~300 m

Slicing

roller-tube

interface

collagen

Culture (for months)


Functional genomics a new addition to chinese medicine research development

Advantages of Brain Slice Culture

  • Organotypic architecture

  • 3-dimensional connectivity

  • neuron-glia interactions

  • tissue-specific transport, ion diffusion systems

For drug development:

  • Easy manipulation of defined neurons and synapses

  • NO blood-brain barrier— better accessment

  • Higher throughput for screening


Functional genomics a new addition to chinese medicine research development

Application of Brain Slice Culture

  • Neural development, Neurogenesis

  • Brain damage models

+ OGD

  • stoke/ischemia

  • ––oxygen/glucose deprivation (OGD);

  • or excitotoxicity

PI: +

OGD

  • epilepsy

PI: 

  • Alzheimer‘s disease

  • infection-related (e.g. HIV) neurotoxicity; traumatic injury; etc.

  • Neuroprotection, neurorepair mechanism

  • Neuropharmacology—protective & toxic chemicals

Chinese Medicine —neuroprotection and repair


Functional genomics a new addition to chinese medicine research development

Functional genomics tools facilitate research & development of Chinese medicine

New drug screening

Organisms

(Mouse, Rat, etc.)

Organs

(Organotypic slice culture)

Phenotype

Cells

(Cell microarray)

Chinese medicine

Mechanism

DNA

Transcript

Protein


Functional genomics a new addition to chinese medicine research development

Genomics

Diseases

Pharmacology

Phytochemistry

etc.

Chinese Medicine


Functional genomics a new addition to chinese medicine research development

Acknowledgements

Department director

Prof. Hans Lehrach

Collaborators:

Leibniz Institute for Neurobiology:

Klaus Reymann

Ulrich Schröder

Automation & Technology

Wilfried Nietfeld

Cell Microarray

Michal Janitz

Dominique Vanhecke

Andrea Fiebitz

Charite Medical Center:

Johannes Vogt

Leslie Schlüter

German Resource Center for Genome Research:

Florian Wagner

Human Chromosome 21

Marie-Laure Yaspo

Hans-Jörg Warnatz

歡迎開展中醫藥研究合作,

共同探索中醫藥發展新路。

Max-Planck-Institute for Molecular Genetics

Berlin, Germanyhttp://www.molgen.mpg.de/


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