TRANSGENIC TECHNOLOGIES: Gene-targeting

Reverse Genetics

Wild-type Bmp7 -/-

Forward Genetics

Phenotype Gene

orMutations First Molecular Analysis

Second

Reverse Genetics

Gene Phenotype

orMolecular Analysis First Mutations Second

"Model" Organisms in Biology

What allows us to use them?1. All organisms share similar cellular machinery

2. All animals use this machinery in similar ways to direct

embryonic development

How about behaviors?-Species-specificity

-Phonotypical homologues

-Pharmacological homologues

-Reverse genetic homologues

Mouse model of drowning?

Transgenic mice

• Transgenic mice

• Knockout mice

• Knockdown

• Gene-trap

Transgenic mice

Early 1980,

Mrio Capecchi: Homologous recombination

in fibroblasts

Martin Evans: Isolation of ES cells

1987, Mario Capecchi and Kirk Thomas

First gene-targeting in ES cells

1989, Rudolf Jaenisch in MIT

Generation of knockout mice, beta-2

microglobulin

In Korea,

1993, H3 ES cell line

1995, IP3K, PLCb1 KO mice born

Gene-knockout mice

ES Cells into

Blastocysts

Strain with recessive coat color

neo

neo

ES Cell

Transfection

of targeting

vector

Germ-line

Germ-line

F1

Chimera

Embryo

Transfer

to Uterus

Foster

F2

Timeline for the generation

of ES cell-derived mice

Introduce

targeting

vector into

ES cells

Identify

homologous

recombinants

by DNA

analysis

Identify mouse

Chimeras with

high ES cell

contribution Germline transmissionBegin

analysis

0 2 4 8 10 126

Drug

selection

Colony growth

and expansion

Inject

clones

into

blastocysts

Sexual

maturation

of chimeras

Identify

male and

female

heterozygotes

Sexual

maturation of

heterozygotes

Identify

homozygotes

EMBRYONIC STEM CELLS

ADVANTAGE:

-Totipotency

-Manipulation in Culture

(screening rare events)e.g. Lotto, 1/8 x 106 vs. homologous recombination106~8

USE:

-Generation of Transgenic Mice

-Gene Targeting-specific Gene Manipulation

-Gene Trap-Random Gene Mutation

Early mouse development

From Sedivy &

Joyner “Gene

Targeting” 1992

Derivation of Embryonic Stem Cells

From old ES cells

1. Plating of ~100 ES cells per

10cm dish

2. Cuture for 7-10 days with LIF

3. Isolation of

single cell-derived colonies

4. Karyotyping (40XY)

5. Characterization of

germ-line competence

From ICM

1. Isolation of PND 3.5

blastocysts

2. Culture on embryonic

fibroblasts (EF)

3. Isolation of hatched ICM

4. Trypsinization of ICM and

culture on EF

5. Isolation of ES colonies

6. Karyotyping (40XY)

7. Characterization of

germ-line competence

ES or Embryonic stem cells:

Blastocyst-stage cellsthat have been coaxedand coddled intogrowing in culture

ICM

Normal

C57BL/6

Blastocyst

(black)

ES cells

129/SvJae

(Blackagouti)

agouti black

Making chimera with Morula

A mouse with“3 parents”

♂ chimera ♀ FvB

Chimeras from foster mother Backcross with female having

recessive coat color

The first chimera and germ-line

transmission in Korea!!

• Multi-copy genes in the mouse genome

• Genes unexpressed in mouse

Genes unable to be KO

Serotonin-N-acetyltransferase

A1 a1G

410 bp

9.4

6.6

4.4

Generation of targeting vector: Overall Procedure

Isolation of cDNA

• RT-PCR

Isolation of genomic clones

• Phage library

• Bacterial Artificial Chromosome (BAC)

• Genomic PCR

Restriction mapping of genomic clones

• Axon mapping

Vector construction

• Insertion of neo cassette into the target exon

• Attachment of negative selection marker

Neo

Gene Targeting By Homologous Recombination

Neo TK Plasmid

sequence

Wild-type locus

Targeted locus

probe

probe

Targeting Vector

XX

XX

1. Homologous arm x 2

2. Neo marker

3. TK marker

Generation of targeting vector :

Which exon should be targeted to make null

mutation?

Preferred

- First coding exon

- Functionally important exon

Avoided

- Exon can be skipped by alternative splicing

- Number of nucleotides with a multiple of 3

Exon mapping

CCACATTgtn---------------------agCAGAA

...CCACATTCAGAA...

...ProHisSerGlu...

Splice donor acceptor

CCACATTgtn---------------------agcagAA

...CCA CAT TAA...

...Pro His STOP

Exon mapping

1 3 4 52 6

Start Stop

1 3.1 4 52 6

Start Stop

3.2

Start

1 1 3 22 3

Start Stop

1 3 4 52 6

Start Stop

1 3.1 4 52 6

Start Stop

3.2

Start

1 1 3 22 3

Start Stop

neo

neo

neo

Positive selection:

ES cells that have the targeting vector: neo resistant

Negative selection:

Select against ES cells with random vector integration

sensitive to anti-herpes drugs, FIAU, gancyclovir

3 HSVtk1 neo

random

31 neo

recombinant

Selected out

alive

Probe 1 Probe 2

+/+ +/- random +/+ +/- random

Molecular screen:

Eliminates random integrants without HSVtk

Use flanking sequences

31 neoProbe 1

31 2

targeted allele

endogenous allele

Probe 2

Disrupted

locus

Wild type

locus

Targeting

Vector

B EHB B HS H H BES

8.6kb

B E BES

B EB HS H H BES

12.6kb

T K

H

Neo

Neo

H

H

PGK

F1

B1

B1

12.6

8.6

23.13

6.557

124

80

209

+/+ +/- -/-(kDa)

(kb)

Gene-targeting of a1G T-type channels in Mice

+/+ -/-

Factors influencing targeting efficiency

• isogenic DNA (perfect homology)10-25 fold van Deursen J, Wieringa B. Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors. Nucleic Acids Res. 20:3815-20, 1992.

• size of region of homologyexponential relationshipCapecchi MR. Altering the genome by homologous recombination.Science. 244:1288-9, 1989

• robust screen!Positive controlsRun Repeat masker

• intrinsic features of the locus

-Recombination hot spots

Modified KO technologies

• Knock-in

• Gene-trap

• Conditional KO

-Region-specific

-Time-specific

Knock-in technology for analysis of gene isotype function

1 3 4 52 6

Start Stop

neoC-DNA pA

e.g. PLCbeta1 locus

PLCbeta4 cDNA

Gene traps

Exon trap : Insertion into endogenous exon

-Neo-pA

Intron trap :

Splice acceptor-IRES-Neo-pA

Poly-A trap :

Promotor-Neo-Splice donor

Gene traps are alternatives to knockouts

Splice acceptor

Electroporate ES cells

Select for neoR

identify trapped gene

by 5’ RACE, sequencing

Trapped gene

(~random)

promoter exons

splicing

transcripts

Select genes from gene trap library

for blastocyst injection

lacZ pA Promoter neoR pA

You don’t need to make gene-trap!

Manitoba Gene Trap Database Geoff Hicks http://www.escells.ca/

Soriano Gene Trap Lines http://www.fhcrc.org/labs/soriano/research/trap.html Omnibank (Lexicon) knockout clones - library http://www.lexicon genetics.com/omnibank/omnibank_ebiology.htm CMHD: Centre for modeling human disease, Mt. Sinai Hospital, Toronto, Canada http://cmhd.mshri.on.ca/sub/genetrap/paradigm.htm Bay area resource of Mouse Mutations in Secreted and Membrane Proteins http://ist-socrates.berkeley.edu/~skarnes/resource.html German Mouse Gene Trap Database http://tikus.gsf.de/index.html

International Gene Trap Consortium

(gateway to all other databases) http://www.genetrap.org/

Conditional Ko by CRE-lox system

CREGFAP promotor

Neuron

glia

e.g. Astrocyte specific KO

Conditional Ko by tet system

CRE

How to knockout a gene in adult stage by using

tet-off (tetR) system?

Conditional Ko by CRE-lox system

Other Reverse Genetic Approaches

• Site-directed mutagenesis

• RNAi (siRNA or shRNA)

• Chemicals (Chemical Genetics)

Site-directed mutagenesis

Point mutations, domain replacement

Gene Replacement

RNA Interference

Method 1 Method 2 Method 3

Mechanism of RNAi

Forward and Reverse "Chemical Genetics"