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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

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

~ 3 billion nucleotides

~ contain 20–25,000 protein-coding genes

Nature (2001)

Science (2001)

Nature (2004)


New focus in post-genomic era:

Functional investigation of DNA sequences

Genome

Phenotype

Environment


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.


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)


Regulation networks: (Group Nyarsik)

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


…… (Group Nyarsik)

Chinese medicine

Genome

Phenotype

Environment

DNA

Transcript

Protein

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

Functional genomics tools


…… (Group Nyarsik)

Genome

Phenotype

Environment

DNA

Transcript

Protein

Therapeutic mechanism

Chinese medicine

Diseases

Functional genomics tools


New drugs (Group Nyarsik)

  • 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


Principle of Cell Microarray (Group Nyarsik)

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


Apoptosis (Group Nyarsik)

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


Protein co-localization with 9 organelles (Group Nyarsik)

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


Intracellular trafficking on cell array (Group Nyarsik)

6xHis-Chr21 Compartments Merged

gene: KCNE2

1. Golgi vesicles

Golgi

2.Plasma membrane

Mito

3. Plasma membrane & Lysosome/Endosome

Lysosome


Nucleus (M phase) (Group Nyarsik)

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)


Cell death (Group Nyarsik)detection

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 )


siLaminA/C (Group Nyarsik)

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


Subcellular (Group Nyarsik)

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


Transfected Cell (Group Nyarsik)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


New drugs (Group Nyarsik)

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


Organotypic Brain Slice Culture — Hippocampus (Group Nyarsik)

Brain

Dissection

P0-P9

hippocampus

~300 m

Slicing

roller-tube

interface

collagen

Culture (for months)


Advantages of Brain Slice Culture (Group Nyarsik)

  • 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


Application of Brain Slice Culture (Group Nyarsik)

  • 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 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


Genomics of Chinese medicine

Diseases

Pharmacology

Phytochemistry

etc.

Chinese Medicine


Acknowledgements of Chinese medicine

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, Germany http://www.molgen.mpg.de/


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