숙명여자대학교 / 생명과학과박종훈

Biotechnology 의 최신 동향

1. Genome: > 75 yrs

2. Genomics : T. Roderick in 1986

Structural genomics Functional genomics

Functional GenomicsFunctional Genomics

Post-Genome EraPost-Genome Era

Unknown Genes

Known Genes

Functional Genomics cDNA Microarray Proteomics Bioinformatics

Finding of novel functional genes

Disease Drug target

Human Genes 36,655

In vovo functional genomics?In vovo functional genomics?

Germ

line m

odificatio

n

Isolate Blastocyst

Derive ES cell line

Gene Targeting

Inject intoBlastocyst

Transfer to surrogate female

Chimera

Host blastocyst derived progeny ES cell-derived progeny

Polycystic Kidney Disease (PKD)

Autosomal Dominant Polycystic Kidney Disease (ADPKD)

PKD1 - chromosome 16p 13.3PKD2 - chromosome 4p21-q23Non-PKD1-PKD2

PKHD1 - chromosome 6p 21.1-p12

Autosomal Recessive PolycysticKidney Disease (ARPKD)

Structure of PKD1

Structure of PKD2

Characterization of mouse pkd2 and human PKD2 gene

pkd1 PKD2

Physical spanning: ~42 kb 68 kb

Transcript Size: 5.2 kb 5.4 kb

ORF: ~2.9 kb 2.9 kb

Amino Acids: 966 968

Identical at Nucleotide level: ~87%

Homologousat AA level: 90.8%

Functional Domain: conserved

Location: Chr. 5 Chr.4q21-23

Expression Level: Five fold greater in High in human ovary, mouse kidney than fetal and adult kidney, other organs testis, fetal lung, and intestine

Targeting of the Pkd2 Locus

The Pkd2 Null Mutation

EF-handDomain

Primers for Mutagenesis

Constructs of PKD2 C-terminal region

6th TM

PGW2B8-1

PGW3

PGW4

PGW5

PGW5

EF-handPKD2

-X -Z

6th TM

B8-1

EF-hand

PKD2

PGW6-X -Z

A

BG

ST

B8-1(

W)

B8-1(

M)

M GS

T PGW

6(W

)PG

W6(

-Z)

PGW

6(-X

)

20648

35

29

20

206

48

35

29

20

C

What is cDNA microarray?What is cDNA microarray?

SamplePreparation

BiologicalQuestion

MicroarrayLife Cycle

MicroarrayReaction

MicroarrayDetection

Data Analysis& Modeling

DNA Chip 이란 무엇인가 ? DNA Chip 이란 무엇인가 ?

생명과학 +

전자 , 기계공학

핵심기술 : 1) DNA Mcroarray 2) Biochip 3) Lab on a Chip

DNA Chip

GeneFilters MicroarraysGeneFilters Microarrays

a. Spotting Format

b. Control

c. Induced Sample

Selected DNA MicroarraySelected DNA Microarray

Photochemical technology Ink-Jet technology Glass Slide

1. Gene Expression Analysis1. Gene Expression Analysis

Technology AccessTechnology Access

Target preparation Probe preparaion Data analysis

cDNA Microarray Analysis of DNA copy-number changescDNA Microarray Analysis of DNA copy-number changes

a

b c

2. Affymetrix Chips2. Affymetrix Chips

GeneChip Probe Array Synthesis ProcessGeneChip Probe Array Synthesis Process

1. Light2. Deprotected area Activation3. Chemical coupling4. New Mask5. Coupling6. Repeating

PROCESS :

Sequence Analysis ArraySequence Analysis Array

Genotyping ArraysGenotyping Arrays

Maskless Array DNA Chip TechnologyMaskless Array DNA Chip Technology

UV Light SourceFilter

Digital Micromirror Array Condenser

From Computer

From DNASynthesizer Projection System

DNA Chipbeing written

3. Nanogen DNA Chip3. Nanogen DNA Chip

Semiconductor Microchip Permeation Layer

Electronic Addressing of Nanogen DNA ChipElectronic Addressing of Nanogen DNA Chip

4. Hightlights of Clinical Micro Sensors DNA Chip4. Hightlights of Clinical Micro Sensors DNA Chip

CMS Technology

Handheld DNA Chip Reader