1. How we can achieve disruption of the genes essential for cell viability in DT40 cells? Tackling essential genes; tagging Minoru Takata Laboratory of DNA Damage Signaling, Department of Late Effect Studies, Radiation Biology Center, Kyoto University

2. Make a conditional knockout! 1.Testing an essential gene 2.Essential combination (synthetic lethality) 3.Deleting a gene in a mutant that shows low targeting frequency (e.g. HR mutants)

3. Generation of Conditional Mutant Cells Wild-type Heterozygous Homozygous Dead Homozygous Alive 1st allele 2nd allele Dead Random integration of A conditional gene 2nd allele Heterozygous Off

4. 1.Tet off system based on integration of two plasmids cf. promoter hijacking strategy 2.Auxin degron system -Use of bicistronic vector system -In frame knock-in of the degron 3. Inducible Cre-loxP system: - use of an expression vector incorporating loxP sites - knock-in of the loxP sites (with an epitope tag)

5. Tet off system

7. Generation of Conditional Mutant Cells Wild-type Heterozygous Homozygous Dead Homozygous Alive +hRad51 1st allele 2nd allele Dead Random integration of tTA and Tet-hRad51 expression vectors 2nd allele Heterozygous +hRad51 Tet Rad51-/- cells were created following this strategy with sequential transfection. Tet, tetracycline.

8. Regulation of hRad51 protein expression in conditional Rad51 -/- DT40 Wt DT40 RAD51+/- RAD51+/- tet-hRAD51 RAD51-/- tet-hRAD51 Human B cell Ramos 0369121815 hrs Time after addition of tetracyclin Human Chicken Human hRad51 migrates a bit faster than chicken counterpart as shown here. Expression of hRad51 can be rapidly turned off upon addition of tet into culture medium.

9. Chromosome breaks in Rad51 -/- DT40 Isochromatid breaks are observed frequently in the absence of Rad51, suggesting spontaneous occurrence of DSB during replication and its repair by HR pathway. Isochromatid break Chromatid break

10. Auxin degron system

11. You can buy this vector …though a bit expensive…

12. TIR1 is an E3 ligase from plants. Auxin degron (AID) interacts with TIR1 containing SCF complex in the presence of the plant hormone auxin. Nat Methods. 2009 Dec;6(12):917-22. Epub 2009 Nov 15.An auxin-based degron system for the rapid depletion of proteins in nonplant cells.

13. AID system can be used in two ways in DT40 1.Expression of OsTIR1 and degron-fused protein of interest from the bicistronic plasmid vector system (random integration) 2. Knock-in of the degron at the termination codon of the gene of interest

14. Example 1 Expression of degron-fused FANCD2 in fancd2 knockout cells Aim – Testing the system – To make doubleknockouts in fancd2 background

15. 500  M IAA - 153060120min fancd2 + AID-EGFP-chFancD2 WT AID-GFP-chFancD2 anti-FANCD2 fancd2 + AID-chFancD2 WT 500  M IAA - 153060120min AID-chFancD2 anti-FANCD2 D2 GFP AID D2 AID D2

16. IAA -, Cisplatin - IAA +, Cisplatin - IAA -, Cisplatin + IAA +, Cisplatin + 500  M IAA, 2hr ↓ 1.0  M Cisplatin, 48hr + AID-EGFP-chFancD2 WT + AID-chFancD2 WT 0 20 40 60 80 100 120 WTfancd2#1#2#3#1#2#3

17. IAA -, Cisplatin - IAA +, Cisplatin - IAA -, Cisplatin + IAA +, Cisplatin + 500  M IAA, 2hr ↓ 1.0  M Cisplatin, 48hr + AID-EGFP-chFancD2 WT + AID-chFancD2 WT 0 20 40 60 80 100 120 WTfancd2#1#2#3#1#2#3

18. IAA -, Cisplatin - IAA +, Cisplatin - IAA -, Cisplatin + IAA +, Cisplatin + 500  M IAA, 2hr ↓ 1.0  M Cisplatin, 48hr + AID-EGFP-chFancD2 WT + AID-chFancD2 WT 0 20 40 60 80 100 120 WTfancd2#1#2#3#1#2#3

19. Example 2 Knock-in of degron at the termination codon of Ino80 gene Aim – Testing the method – To test the chromatin remodeling factor in DNA damage response and replication – To bypass the possibility of lethality and the secondary effects through transcriptional regulation

20. Knock-in strategy of Auxin-induced degron into the Ino80 C-terminus in wild type cells expressing OsTIR1 ch Ino80 locus Knock-in vector * termination codon Probe HR events without Degron knock-in HR events with Degron knock-in

21. 0153060120180 Ino80-degron FancD2 min Auxin addition

22. Inducible Cre-ER/loxP system

23. Example 1 To make double knockout of Xrcc3 and Rad52 Aim – To elucidate the genetic relationship between the two

24. Rad52 and Rad51 paralogs are important regulator of Rad51 function in homologous recombination (HR). Yeast Rad52 is critical for HR while mammalian or chicken Rad52 has a minimal function. There are only two Rad51 paralogs (Rad55 and 57), while chicken or human Rad51 paralog family has five members (Xrcc2/3, Rad51B/C/D).

25. What is the relationship between Rad52 and Rad51 paralogs in vertebrates? It is impossible to disrupt a gene in Rad51 paralog mutant. Xrcc3 targeting in rad52 mutant failed. Synthetic lethality?

26. Hsp90 Cre-ER OH-TAM GFP IRES loxP Cre-ER loxP PCMV Off! CytoplasmNucleus XRCC3

27. Cre-ER P  -actin neo XRCC3 GFP IRES loxP P CMV neo Co-transfection to RAD52-/- / XRCC3+/- Random integration Identify clones by :Anti-ER western FACS for GFP Genomic Southern and FACS after addition of OH-TAM Strategy for Cre-ER/loxP system

28. rad52 XRCC3 +/- rad52XRCC3 +/- hXRCC3 + rad52 xrcc3 hXRCC3 + hRAD51 + wild-type DT40 rad52 xrcc3 hXRCC3 - rad52 xrcc3 hXRCC3 - hRAD51 + + OH-TAM rad52 xrcc3 hXRCC3 +

29. IRES hXRCC3 EGFPCMV XR3 SP6 CMV A B 0 & XR3 CMV & SP6 246101224488 Hours after OH-TAM addition primerpair loxP Inducible Cre-mediated deletion of hXRCC3 expression cassette

30. 1.9 14.9 2.3 1.8 1.9 0 hr 24 hrs 48 hrs 72 hrs Wild-type RAD52-/- XRCC3+/- +hXRCC3 +IRES-GFP +Cre-ER Time after OH-TAM addition GFP fluorescence Cell number Numerals represent mean fluorescence intensity FACS analysis of GFP expression

31. TAM,5 days RAD52-/-/XRCC3-/-RAD52-/-/XRCC3+/- Forward scatter (cell size) PI 012345 0 20 40 60 80 100 %viability Days after TAM A B

32. Overlapping roles of Rad52 and XRCC3? - No phenotype in rad52  - Some defects in xrcc3  - Synthetic lethality of rad52  xrcc3  - Over-expression of Rad51 suppresses lethality of the double mutant

33. Example 2. Making ATRIP conditional knockout and knock-in of GFP at the c-terminus of ATRIP Aim – To elucidate the role of ATR/ATRIP kinase complex in the Fanconi anemia pathway activation

34. FancI phosphorylation acts as a molecular switch in the FA pathway Ub D2 I Ub D2 I Ub D2 I Ub PPP M B 24 C E F G L A 100

35. ATRIP RPA ATR 9-1-1 complex 17 9 TopBP1 P M N R ATM AB DSB activates ATMATR is activated by ssDNA-RPA Upstream kinase activation in the DNA damage response end resection fork collapse

36. Chicken ATRIP locus (chr 12) bsr GFP * * probe Knock-in/conditional vector termination FRT Knock-out vector Step 1: 1 st allele knock-out in DT40Cre1 expressing Cre-ER. Step 2: 2 nd allele knock-in. To facilitate detection of ATIRP expression, GFP tag was also knocked-in at the termination codon of ATRIP. Step 3: The bsr cassette removed by Flp expression. Step 4: Addition of tamoxifen to activate Cre recombinase. ATRIP gene targeting LoxP

37. 51 ％ 6％6％ 26 ％ 7％7％ 62 ％ 24 ％ DT40Cre1 56 ％ 8％8％ 24 ％ TAM 0h 24 h 31 ％ 11 ％ 20 ％ 48 h DNA content (PI) BrdU uptake ATRIP conditional knockout cells ATRIP-GFP Parental DT40Cre1 ATRIP conditional knockout cells +-+-+-+- 48h24h0h TAM MMC 100ng/ml A B

38. FancI FancD2 Parental DT40Cre1 ATRIP conditional knockout cells Chk1 +-+-+-+- 48240 hrs after TAM MMC 100ng/ml ATRIP is required for the FA pathway activation

39. Summary FancI phosphorylation acts as a molecular switch in the FA pathway Ub D2 I Ub D2 I Ub D2 I Ub PPP M B 24 C E F G L A 100

40. meritsdemerits Tet systemStraight forwardLeaky expression Slow reduction of expression Two plasmids based system Promoter hijacking Inducible Cre-loxPNo leakiness in excised cells Not 100% efficient Complicated vector construction DT40Cre1 Or make your own parental cells Auxin degron systemQuick reduction of expression The system not completely optimized Single plasmid based Or knock-in of degron