Transcript Gene targeting: vector design and construction

Gene targeting:
vector design and construction
Minoru Takata
Radiation 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).
.
Let’s start! –collect information
• 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! – design of the vector
• 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).
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
encoding the critical domain
Sal Sac Sac
*
my gene of interest
term
Sac
No NotI and BamHI in the genomic region (at least in the database)
encoding the critical domain
Sal Sac Sac
*
my gene of interest
term
Sac
No NotI and BamHI in the genomic region (at least in the database)
encoding the critical domain
Sal Sac Sac
Sal
*
B
3.7 kb
term
0.8 kb probe
Sac
NotI
B
4.6 kb
No NotI and BamHI in the genomic region (at least in the database)
encoding the critical domain
Sal Sac Sac
Sal
*
B
term
NotI
B
3.7 kb
4.6 kb
NotI
Sal
pBluescript
0.8 kb probe
Sac
No NotI and BamHI in the genomic region (at least in the database)
encoding the critical domain
Sal Sac Sac
Sal
*
B
term
NotI
B
3.7 kb
B
bsr
4.6 kb
Sac
B
NotI
Sal
pBluescript
0.8 kb probe
Sac
No NotI and BamHI in the genomic region (at least in the database)
encoding the critical domain
Sal Sac Sac
Sal
*
3.7 kb
Sac
bsr
pBluescript
term
4.6 kb
0.8 kb probe
Sac
NotI
No NotI and BamHI in the genomic region (at least in the database)
encoding the critical domain
Sal Sac Sac
Sal
*
3.7 kb
Sac
bsr
pBluescript
term
4.6 kb
0.8 kb probe
Sac
NotI
encoding the critical domain
Sal Sac Sac
Sal
*
3.7 kb
Sac
bsr
term
4.6 kb
linearized!
pBluescript
0.8 kb probe
Sac
NotI
~15 kb
Sal Sac Sac
*
term
Sac
0.8 kb probe
Sac
targeting vector
bsr
~5.5 kb
Sac
Sal Sac Sac
bsr
Sac
targeted locus
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.
Generation of FANCG deficient cells
A.
~20kb
E
Xh Xb
BB B
~15kb
Xh Xb
B. Southern blot
E
his
chFANCG locus
Targeting vector
E
Targeted locus
C. RT-PCR
WT
WT fancg
(kb)
E
E
his
probe
E
B
fancg
FANCG
20
15
RAD51
Only single allele in case of this, It is on Z chromosome, which is syntenic to human Chromosome 9.