Gene targeting: vector design and construction

Minoru TakataRadiation Biology Center,

Kyoto University

Components of targeting vector

• Vector backbone– pBluescript (cheap!), TOPO-XL (Invitrogen)

• Left homology arm (1-4kb)• Resistance gene cassettes

– hisD, bsr, puro, neo, hygro, ecoGPT – flanked with BamHI sites– Floxed (or flanked with FRT sites) versions

available• Right homology arm (1-4kb)

Points to be considered(1):• How many alleles in the gene of your interest?

– Chromosome No.2– Chromosome Z– You can recycle the resistance gene cassette by using Cre-loxp

system

• Which genomic region you would like to delete by replacing resistance gene cassettes? – Functionally important region should be deleted– Destroying the exons? (deletion at the middle of the exon)– Size of the genomic region to be deleted. Maybe not good if too

huge

• Length of the arms. – Longer the better. L arm +R arm > ~5-6 kb– One arm could be short but should be at least 1kb.

Points to be considered(2):• Mapping of BamHI sites (and BglII sites).

– You may need to replace a resistance gene cassette with another one. They are flanked by BamHI.

• Vector linearization site. – A cutting site at the end of the homology arm is probably

good.– PvuI in pBS (two sites in the vector) : still OK– Unique site in Amp ; still OK– No linearization results in fewer colonies but could be OK

Points to be considered(3):• Screening strategy.

– PCR? Long arms not appropriate for PCR screening.– Southern? Probe (must not hybridize with the arms) and

restriction sites are needed.

• Complementation– expression vectors. CMV promoter works fine; neo, puro,

hygro, zeo available, e.g. pcDNA3.1– chicken beta-actin promoter may be good to achieve

higher expression levels (pApuro vector)..

• Get sequence of your gene of interest.

• How can you be sure you are deleting the chicken ortholog of your gene of interest?– Degree of homology may depend on the gene. – look at the genes surrounding your gene. If you find synteny, it is

OK.

• Which chromosome? Three copies of chromosome #2; single copy of chromosome Z in DT40, which was derived from female chicken.

• Analyze/map genomic structure (exon-intron) of your gene of interest. – don’t trust the database too much! There could be mistakes,

differences (polymorphisms) and SNPs.

Let’s start! –collect information

• Design PCR primers – incorporate appropriate sites for cloning – don’t forget to add 2-3 nucleotides to the ends to ensure

complete digestion

• For more complicated construction (knock-in etc), consider using Multi-mutagenesis kit (Stratagene).

Let’s start! – design of the vector

Cloning!• Get left and right arm fragments by LA-PCR

– Clean-up products and then digest them• prepare vector backbone

– Digest pBS by appropriate enzymes (e.g. NotI and SalI).

• Ligation and transformation– Try three fragment ligation using blue white selection– if failed then proceed one by one

• Insert resistance gene cassettes to BamHI site– Isolate clones with both orientations, since the

orientation may affect efficiency of targeting

*Sal Sac Sac Sac

encoding the critical domain

term

my gene of interest

*Sal Sac Sac Sac

encoding the critical domain

term

No NotI and BamHI in the genomic region (at least in the database)

my gene of interest

*Sal

3.7 kb 4.6 kb

0.8 kb probeSac Sac Sac

encoding the critical domain

term

No NotI and BamHI in the genomic region (at least in the database)

Sal B B NotI

*Sal

3.7 kb 4.6 kb

0.8 kb probeSac Sac Sac

encoding the critical domain

term

No NotI and BamHI in the genomic region (at least in the database)

Sal B B NotI

pBluescript

Sal NotI

*Sal

3.7 kb 4.6 kb

0.8 kb probeSac Sac Sac

encoding the critical domain

term

No NotI and BamHI in the genomic region (at least in the database)

Sal B B NotI

bsrbsrSac

B B

pBluescript

Sal NotI

*Sal

3.7 kb 4.6 kb

0.8 kb probeSac Sac Sac

encoding the critical domain

term

No NotI and BamHI in the genomic region (at least in the database)

bsrbsrSac

pBluescript

Sal NotI

*Sal

3.7 kb 4.6 kb

0.8 kb probeSac Sac Sac

encoding the critical domain

term

No NotI and BamHI in the genomic region (at least in the database)

bsrbsrSac

pBluescript

Sal NotI

*Sal

3.7 kb 4.6 kb

0.8 kb probeSac Sac Sac

encoding the critical domain

term

bsrbsrSac

pBluescript

Sal NotI

linearized!

*Sal0.8 kb probe

Sac Sac Sacterm

bsrbsrSac

bsrbsrSac

targeting vector

targeted locusSal Sac Sac Sac

~15 kb

~5.5 kb

Gene targeting of tyrosine kinase Lyn

My first DT40 knockout. Unfortunately, there were three alleles…

There are three band, since there are three alleles in Lyn locus.

B. Southern blot

A.

B B BBXbXh

Targeting vectorhisE

E E

~20kb

hisEE

~15kb

chFANCG locusprobe

E Targeted locusXbXh

C. RT-PCR

WT fancg

(kb)

1520

FANCG

RAD51

WT fancg

Generation of FANCG deficient cells

Only single allele in case of this, It is on Z chromosome, which is syntenic to human Chromosome 9.