molecular genetics (분자유전학 -...

Molecular genetics (분자유전학)

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2010년 2학기

Mouse mutagenesis

Mouse mutagenesis

Reverse genetics approach

• Gene-driven approach

• well known sequence and structure of a target gene

• Tools

- Gene targeting

- gene-trap mutagenesis

- RNAi mediated knockdown

- ENU in vitro

Generation of KO mouse mutants by gene targeting in ES cells

- Establishment of ES cell line

- Germline transmission in chimeric mice

- Homologous recombination

Establishment of ES cells




ES cell mutagenesis and chimera production

Target vector construction

- Establishment of ES cell line- Germline transmission in chimeric mice


- ET- Recombineering

Web-based resources for mouse mutagenesis

Conditional knockout

Cre-loxP system

Chromosomal engineering

Principle of conditional gene targeting using the Cre/loxP recombination system.

Establishment of ES cells




ES cell mutagenesis and chimera production

Target vector construction

- Establishment of ES cell line- Germline transmission in chimeric mice


- ET- Recombineering

Screen genomic library for genomic clone

Select for insertion event

Isolate surviving clones and screen DNA

Blastocyst Injection

Chimeric mice

Breed heterozygous mice to produce homozygous mouses

Transfection by electroporation

Prepare Primary Embryonic Fibroblasts

Grow ES cells on fibroblast feeder layer

Construction of targeting vector

Cre recombinase- consists of 4 subunits and two domains

- larger carboxyl (C-terminal) domain catalytic site

: similar in structure to the domain in the Integrase family of enzymes isolated

from l phage

- smaller amino (N-terminal) domain

- recombination of DNA between specific sites in a DNA molecule (LoxP)

Lox P site (from phage 1)

13bp 8bp 13bp

ATAACTTCGTATA -GCATACAT-TATACGAAGTTAT

Cre-Lox recombination

A.

Cre

LoxP LoxPGene Insert

+

Insert

Excision

Integration



B.

inverstion Cre

FLP-Frt recombination

- Site-directed recombination technology used to manipulate an organism's DNA under

controlled conditions in vivo and in vitro.

34bp long FRT site sequence

: derived from the S. cerevisiae 2mm DNA plasmid,

a selfish genetic elements present in about 60 copies/cell

Flipase(FLP) binding site

5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3'.

FRT FRT

FRT FRT

FRT FRT

+

A. Inversion B. Excision/Integration

Recombineering

Utilizes the efficient homologous recombination functions encoded by λphage or cryptic phage Rac to perform DNA manipulation in E. coli.

Bacteriophage λengages in homologous recombination during its lyticcycle, even in a recombination-deficient host, through expression of its red genes (1968)

E. Coli has multiple recombination pathways.

• RecBCD pathway

• RecE pathway

• RecF pathway

Bacteriophage λ

• Red recombination system

IntegrationExcision

HomologousRecombination

Early Regulation

DNAReplication

?

LateRegulation

Lysis

Head

Tail

Int, xis

Red system:Exo, bet, gam

N, cI, cro, cII

O, P

Nin region:orf, rap

Q

S,R

NuJ-FII

Z-J

λ genes

Recombineering

Three λ phage Red proteins

Gam

• inhibits RecBCD and SbcCD exonuclease activities of E. Coli

• preserving linear dsDNA and allowing it to be used as a substrate for R

Beta

• a single-stranded DNA binding protein that promotes annealing of cDNA strands

• bind to ssDNA longer than 35 bp, and can protect ssDNA overhangs fromsingle-strand nuclease degradationmake it possible to use only about 50 nt of homology for efficient

recombination

Exo

• dsDNA-dependent 5’-3’ exonuclease that processes linear dsDNA and generate a 3’ ssDNA overhang at each end, the substrate that Beta binds.

RecombineeringRecBCD nuclease

Gam

MC1-TK Amp Ori

Exon 3

BAC

Retrieve of genomic DNA of interst into vector

PCR

MC1-TKAmpOri

Electrophoration into SW102

Containing retrieved exon 3

PL452

MC1-TKAmpOri

Electrophoration

into SW102

Insertion of 1st loxP site into retrieved vector

MC1-TKAmpOri

Electrophoration into SW106

expressing Cre recombinase

MC1-TKAmpOri

Re-electrophoration into SW102

: LoxP

MC1-TKAmpOri

NEO

MC1-TKAmpOri

NEO

Insertion of 2nd loxP site into retrieved vector

: LoxP

: Frt

neo

Amp

Runx2 conditional knockout vector

Electrophoration

Embryo transfer

LoxP

Frt

Runx2Exon3

ES cell

Blastocyst

Runx2 Conditional Knockout mouse

105002 3

121100127100137100143100

22500

BstXI

121864122910135750139494140445

Not1

137100

SpeI

122368128474142610 140945

BamHI

128616134616

XbaI

131100 125100140100 134100

Sep 29’ 06

129100 123100

Procedure

1. Transformation of Bac DNA into SW102

2. Retrieving genomic DNA from SW102 containing Bac clone- PCR with long primers- Temp: PL253- Electroporation of linear gap repair plasmid- Selection in Amp LB plate and plasmid DNA extraction- Retransformation of gap repaired plasmid (1ng) into SW102- Confirmation of retrieved gDNA by restriction mapping

3. Targeting the first loxP site into the subcloned plasmid DNA- mini-targeting cassettes for inserting loxP (loxP cassette)

5’ 42bp-hm_RE(6)_loxP(34)_cgacctgcagcctgttga-3’

5’ 48bp-hm(rv)_rv-loxP(34)_gtcgaggctgatcagcga-3’

PCR; 1-2 ng template (PL452), 25 cycles, 1-2 ul DpnI 1h at 37 and gel puri O/N- electroporation of mini-loxP cassette into SW102 containing retrieved gDNA- kanamycin (due to Neo) plate and DNA confirmation- Cre-mediated “pop-out” of this cassette after electroporation into SW106

4. Targeting the second loxP site - mini-targeting cassettes for inserting Frt-Neo-Frt-loxP (2-loxP cassette)

5’ 48bp-hm_gaagttcctattctctagaaagtataggaacttcaggtctgaagaggagttt-3’

5’ 50bp-hm_ataacttcgtatagcatacattatacgaagttatattatgtacctgactg-3’

PCR; 1-2 ng template (PL451), 25 cycles, 1-2 ul DpnI 1h at 37 and gel puri O/N- electroporation of 2-loxP cassette into SW102 containing 1st loxP inserted gDNA

1칸=5kb

3’-6-10kb 정도downstream

Display

View Evidence

Restriction Mapping of BAC

0 100 200 239 Kb120 140 160 18020 40 60 80 220

BH1

SalI

XhoI

NotI

SacII

MluI

ClaI

Recombineering

Bac DNA Confirm

Retrieving of gDNA

Plate test

Bac DNA Prep

The first loxP site

Pop-out floxed neo

The 2nd loxP site

20 30 10 20 30

Primers

1. Targeting the first loxP site into the subcloned plasmid DNAupper: 42bp(hm)_NdeI(6)_loxP(34)_cgacctgcagcctgttga-3’

lower: 48bp(rvhm)_rvloxP(34)_gtcgaggctgatcagcga-3’

(1st-LoxP-Upp)5’-ccggcgggcggcggcagcggctgcgatccgcaggctccagat_catatg_

ataacttcgtataatgtatgctatacgaagttat_cgacctgcagcctgttga-3’

(1st-LoxP-Low)5’-tgccgggaggtaagtgggggcggggggagagcggatctcggggcgacag_

ataacttcgtatagcatacattatacgaagttat_gtcgaggctgatcagcga-3’

2. Targeting the second loxP siteUp:48bp(hm)_gaagttcctattctctagaaagtataggaacttcaggtctgaagaggagttt-3’

Lo:50bp(hm)_ataacttcgtatagcatacattatacgaagttatattatgtacctgactg-3’

(2nd-LoxP-Upp)5’-cattttgcctgggaggaaaagggaatcctgtggagcaacttcggttgg_

gaagttcctattctctagaaagtataggaacttcaggtctgaagaggagttt-3(2nd-LoxP-Low)5’-agttctggctctttcaaggcaaataaagttttcacctctgtttattctga_

ataacttcgtatagcatacattatacgaagttatattatgtacctgactg-3’

5’ probe

RV RV

gDNAfrom Bac

Neo

7.1 kb

8.1 kb

7.1 kb

MC1 TK

MC1 TK

6.0 kb

MC1 TK

Neo

MC1 TK

MC1 TK

RV RV

3.1 kb

4.1

6.0 kb

1.1

MC1 TK

4.0 kb

3.1kb

3.1 kb

3.1 kb

3.1 kb

3.1 kb

MC1 TK

NeoSpeI

SalI

pSK+

SalI

NeoSpeI

5’ probe 3’probe

Conditional target vector

Genomic locus

Targeted allele

BamHI

gDNAfrom Bac

Neo

11 kb

MC1 TK

MC1 TK

5.8 kb

MC1 TK

Neo

MC1 TK

MC1 TK

BamHI

MC1 TK

6.6 kb

7.2 kb

10 kb

10 kb

5.8 kb

4.2 kb

NdeI

NdeI

NdeI

NdeI

NdeI

MC1 TK

NeoNdeI

SalI

pSK+

SalI

NeoNdeI

5’ probe 3’probe

Conditional target vector

Genomic locus

Targeted allele

16.5 kb

NdeI NdeI

4.6 kb

BamHI

12.5 kb

BamHI

1kb

3260 bp

2511bp

5.8 kb

BamHI BamHI

3.0 kb

ZMCTK

2

SpeI/BamHI/XbaIBstXI/NotI Hind3

5’-NotI-homology-Hind3-3’ 5’-Hind3-homology-SpeI-3’

5’ 3’

Plasmid: PL253

Primers:

Cloning of Runx2-Ex2-Retriving vector via three-way ligation

Fragements elusion, restriction digestion and three-way ligation

Sep 29’ 06

Primer A:NotIRunx2-Ex2-Ret-5’-15’-ATAAGCGGCCGCGTCGACGGGAAAGCAAGGAGAGAGAG-3’

Primer B:H3Runx2-Ex2-Ret-5’-2----- 939bp (39+900)5’-GTCAAGCTTACTGCTGCCATGAAATGCTAAGTCTCTTAG-3’

Primer Y:H3Runx2-Ex2-Ret-3’-15’-GATAAACATGGTTGGGAATATTCAAAGGAGA-3’

Primer Z:SpeIRunx2-Ex2-Ret-3’-2----702bp(40+660+2)5’-ACTACTAGTGGTAAGCATCCGTTAAGAGTG-3’

A B Y

2

SalI

BstXI/Not1I

Cloning of Retrieved E3 Vector (RE3 vector) via long primers

R2-Ex2-Ret-5’-Upper (Hm50-NotI-template24)795’-AATCACAAGTATATAAAATAATGTGGAAGGAGTTAGGACGGGAAAGCAAG-CGGCCGC-TCTAGCCTCGAGGCTAGAACTA-3

R2-Ex2-Ret-3’-Lower (rvHm50-SalI-rvtemplate 24)805’-AGTGGTAAGCATCCGTTAAGAGTGTCACTGTGTATGTGATGCCTCACAGC-GTCGAC-tcgtattacaattcactggccgtc-3’

Mc1-TKAmp

BAC

NarI-NheI-ClaI-SalI-KpnI

McTK Amp 5350 bp

Ori

/SpeI/BamHI/Xba

Mini-targeting cassettes for inserting loxP and Frt-neo-Frt-loxP

1st targeting and 2nd targeting step

Primer C:SalIRunx2-Ex2-Mini-5’L-1 (PL452)5’-GTCGTCGACTCAAAGGGGGAGGGCACAGGAGTG-3’ 33mersPrimer D:RIRunx2-Ex2-Mini-5’L-2 (PL452)---323bp(40+240+43)5’-GTCGAATTCAGCAAGGGACCTGTCATGACCCT-3’ 32mersPrimer E:BamHRunx2-Ex2-Mini-5’R-1 (PL452)5’-ATAGGATCCGGCGGATTTCCCGGCTTCTGTGGG-3’ 33mersPrimer F:NotIRunx2-Ex2-Mini-5’R-2 (PL452)---343bp(58+240+45)5’-ATAAGCGGCCGCGTTATGGGGGGAGGCGATTTTGG-3’ 35mersPrimer G:SalRunx2-Ex2-3’L-1 (PL451)5’-GTCGTCGACTCACCCTAATAGTTTCATCTCTCTG-3’ 34mersPrimer H:RIRVRunx2-Ex2-3’L-2 (PL451)---332bp(27+240+65)5’-GTCGAATTCGATATCCTGCTTTCCACCTGACAA-3’ 33mersPrimer I:BamHRunx2-Ex2-3’R-1 (PL451)5’-ATAGGATCCAAAAATACAAGACAGCTAGTAGTCTTCA-3’ 37mersPrimer J:NotRunx2-Ex2-3’R-2 (PL451)---334bp(52+240+42)5’-TAAAGCGGCCGCTACTAGATGCAGGGTCAATGTG-3’ 34mers

MC1-TKAmpOri

PL253

BAC clone

MC1-TKAmpOri

Recombineering

Homologous region 1

Homologous region 2

그림1.

MC1-TKAmpOri

neo

neo

PCR Electrophoration into SW102 containing retrieved gene X

PL452

MC1-TKAmpOri

neo

Electrophoration into SW106expressing Cre recombinase

그림2.

neo Neomycin gene

loxP site

Homologous region 3

Homologous region 4

PCR from PL451neo

그림3.

neo

Electrophoration into SW102 containing first loxP site

neo

neo Neomycin gene

Homologous region 5

Homologous region 6

FRT site

neo pX-FRT

pX-ΔFRT

pX-Δexon

pX-ret : PL253 retrieved vectorpX-neo : PL253 retrieved vector containing 1st neo cassette floxed loxP sitepX-Δneo : PL253 retrieved vector containing 1st loxP sitepX-FRT : PL253 retrieved vector containing 2nd neo cassette franked FRT sitepX-ΔFRT : PL253 retrieved vector deleted 2nd neo cassettepX-Δexon PL253 retrieved vector deleted exon of gene X

A.

B.

그림4.

Chan W et al., NAR 35, e64, 2007

Recombineering of interesting gene

Tools for targeted manipulation of the mouse genome

Van der Weyden, David J Adams and Allan Bradley

Physiol Genomics 11:133-164, 2002

Procedure for the generation of genetically modified mice generated by gene targeting strategies

Examples of some commonly used dominant selectable markers

FIAU, 1(1-2-deoxy-2-fluoro-b-d-arabinofuranosyl)-5-iodouracil

Replacement and insertion type vectors

Replacement vector

Insertion vector

Hit-and-run targeting procedure

“Double replacement” targeting

Tetracycline-responsive regulatory system for transcriptional transactivation

Knock-in strategies

Mechanisms for generating mosaics in vitro and in vivo

Genetic mosaics are individuals that contain cells of more than one genotype,Whereas chimeras refer to individuals that are formed from cells of differentSources.

Genetic mosaic s are generated when mitotic recombination b/w homologousChromosomes occurs during the G2 phase of the cell cycle and the recombinant Chromatids segregate to different daughter cells: (X segregation).

Recombinant chromatids produced in G2 can also segregate to the same daughter cell, (Z segregation). In this case, both daughter cells will be phenotypicallyIndistinguishable from the parent cell or from cells produced by G1 recombination

Z segregation is thus not useful for genetic mosaic analysis.

Mechanisms for generating mosaics in vitro and in vivo

Recombinase target sites

Gene targeting in ES cells

Generation of defined chromosome rearrangements

Nested chromosomal deletions induced with a retroviral vector

Recombineering steps to generate a BAC recombinant

Large-scale dominant and recessive ENU-induced mutation screens

ENU-induced mutagenesis screens for mouse chromosome 11 using

chromosomal engineering technology

The basic trap vectors

Large-scale gene trapping projects

molecular genetics (분자유전학 -...

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