1. Gene targeting: vector design and construction Minoru Takata Radiation Biology Center, Kyoto University

2. 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)

3. 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.

4. 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

5. 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)..

6. 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. – dont trust the database too much! There could be mistakes, differences (polymorphisms) and SNPs. Lets start! –collect information

7. Design PCR primers – incorporate appropriate sites for cloning – dont 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). Lets start! – design of the vector

8. 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

9. * SalSac encoding the critical domain term my gene of interest

10. * SalSac encoding the critical domain term No NotI and BamHI in the genomic region (at least in the database) my gene of interest

11. * Sal 3.7 kb4.6 kb 0.8 kb probe Sac encoding the critical domain term No NotI and BamHI in the genomic region (at least in the database) Sal BB NotI

12. * Sal 3.7 kb4.6 kb 0.8 kb probe Sac encoding the critical domain term No NotI and BamHI in the genomic region (at least in the database) Sal BB NotI pBluescript Sal NotI

13. * Sal 3.7 kb4.6 kb 0.8 kb probe Sac encoding the critical domain term No NotI and BamHI in the genomic region (at least in the database) Sal BB NotI bsr Sac B B pBluescript Sal NotI

14. * Sal 3.7 kb 4.6 kb 0.8 kb probe Sac encoding the critical domain term No NotI and BamHI in the genomic region (at least in the database) bsr Sac pBluescript Sal NotI

15. * Sal 3.7 kb 4.6 kb 0.8 kb probe Sac encoding the critical domain term No NotI and BamHI in the genomic region (at least in the database) bsr Sac pBluescript Sal NotI

16. * Sal 3.7 kb 4.6 kb 0.8 kb probe Sac encoding the critical domain term bsr Sac pBluescript Sal NotI linearized!

17. * Sal 0.8 kb probe Sac term bsr Sac bsr Sac targeting vector targeted locus SalSac ~15 kb ~5.5 kb

18. Gene targeting of tyrosine kinase Lyn My first DT40 knockout. Unfortunately, there were three alleles…

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

20. B.Southern blot A. B B B B Xb Xh Targeting vector his E E E ~20kb his E E ~15kb chFANCG locus probe E Targeted locus Xb Xh C. RT-PCR WT fancg (kb) 15 20 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.