ZFN WORK (JAN 2011- JAN 2013)

1. ZFN WORK(JAN 2011- JAN 2013)

2. STANDARDIZATION TECHNIQUES • PCR • RT PCR • BLOT • PI STAINING • ANNEXIN V • CEL-1/T7E1 ASSAY • HOECHST ASSAY • TRANSFECTION • HIRT EXTRACTION

3. INITIAL EXPERIMENTS WITH SIGMA ZFN PAIRS • Transfection efficiency was good. • RT PCR AND blot of E6 showed down regulation after treatment. Subsequent set of experiments did not show any reproducibility of results. • PI Staining and Annexin V assays showed apoptotic fraction. • Hoechst assay showed chromatin condensation at higher concentration of ZFNs • Cel-1 assay was under standardization

4. SiHa and Hacat Cell lines and PCR amplification for confirmation of E6 (a) SiHa, (b) Hacatcell line, (c) E6 amplification (477 bp) in HPV positive (SiHa Lane 2) and HPV negative (Hacat lane 3) cell line, Lane1 ladder

5. TRANSFECTION EFFICIENCY Co-transfection with GFP showed a good transfection efficiency for the ZFN pairs.

6. RT-PCR IN SIHA AND CASKI E6,E7,GAPDH IN SIHA E6,E7, GAPDH IN CASKI E6 E6 E7 GAPDH

7. WESTERN BLOT 1 2 3 4 E6and β- Actin in SiHa Lane 1 Treated SiHa (ZFN 10 ug) Lane 2 Treated SiHa (ZFN 5 ug) Lane 3 Untreated SiHa Lane 4 untreated SiHa E6 Beta actin

8. T7 E1 ASSAY IN SIHA 1 2 3 4 5 Lane 1 treated ( conc 10 ug) Lane 2 Treated (conc 5 ug) Lane 3 Treated ( conc 2.5 ug) Lane 4 Untreated SiHa Lane 5 Marker Mismatch endonuclease assay Standardization was not done till then.

9. Results of SIHA transfection and Cel I assay

10. Transfection efficiency using Lipofectamine LTX reagent Bright field image Plain GFP fluoresence image – 3 different fields taken SiHa transfection efficiency 30%

11. CEL I assay 1 2 3 4 5 Cut product Lane 5 Ladder Lane 4 Untreated Lane 3 Untreated Lane 4 2 ug treated Lane 1 4 ug treated siha Obtained a cut product in 4 ugzfn treated cells which is not seen in untreated cells. Cel I assay worked but the efficiency of ZFN editing is not very high

12. CEL I assay repeat 1 2 3 4 5 6 7 8 9 10 Cut product 500 bp product of E6 E6*1 300 bp product Lane 1 Ladder Lane 2 Untreated siha without cel I treatment Lane 3 Treated Siha without cel I Lane 4 Treated SiHa (floating cells obtained after treatment) Lane 5- do- Lane 6 4 ug treated siha Lane 7 –do- Lane 8 2ug treated siha Lane 9 -do- Lane 10 Untreated Siha Treated cells (lane 6 and7 4 ug) and floating cells after treatment( Lane 4 and 5) gave a higher signal for the cut product when compared to the untreated

13. There was no reproducibility in result in subsequent round of experiments

14. Annexin V and PI staining in SiHa

17. Hoechst staining and • Hoechst staining showed chromatin condensation at 2 ug Untreated siha Treated siha at 1 ug treated siha at 2 ug

18. TRANSFECTION • There were issues with transfection of siha and caski cell lines. • Several reagents and methods such as- lipofectamine, lipofectamine 2000, lipofectamine LTX, amaxanucleofection, invitrogen electroporation, Calcium phosphate method were used. LTX gave 30-40% efficiency on using plaineGFPvector. • Using LTX, Cel I assay was performed but only a faint cut product was obtained that too it was not reproducible.

19. TRANSFECTION REAGENT • Effectene (qiagen) was standardized for SiHa and CasKi cell line. The reagent increased the efficiency by 50-60% • The ZFNs synthesized by sigma had a flag tag and no fluorescence tag for monitoring transfection efficiency. So a cloning strategy was devised where IRES (clonetech) vector was used

20. Nucleofector I-013 only 2% L 2000 only 5% Electroporation only 3%

21. GFP reverse transfection , ~16% FACS analysis of LTX transfection GFP transfectants

22. SiHa transfection with nucleofector V and program T-030 ~21% transfection efficiency was seen with this program

23. SiHa transfection

24. Modifications done • Plasmids were purified using Qiagen midi kit instead of promega kit (which was used earlier) and transfection was done using Lipofectamine LTX and protocol was followed as per manufacturer’s instructions

25. TRANSFECTION EFFICIENCY IN SiHa and CaSki CELL LINE 70-80% EFFICIENCY IN SIHA CELL LINE ~70% EFFICIENCY IN CASKI CELL LINE

26. Clone 8-IRES containing ZFN 1 and 2 1 2 3 4 5 Lane 1 Ladder Lane 2 ZFN2 digestion (~1.3kb) Lane 3 ZFN1 digestion (~1.2 kb) Lane 4 Uncut vector Lane 5 GFP PCR product 1.5 Kb

27. Restriction digestion and PCR of positive clone 1 2 3 1 2 3 4 5 6 2 kb 2 kb 1 kb Lane 1 ladder Lane 2 blank Lane 3 uncut vector Lane 4 blank Lane 5 blank Lane 6 Cut clone 20 releasing 2kb product Lane 1 ladder Lane 2 negative control Lane 3 clone positive for GFP and ZFN2 PCR

28. 1 2 3 4 5 10 kb 8 kb 6 kb Lane 1 ladder Lane 2 clone 18 ( gfp and ZFN2) Lane 3 clone 19 (gfp ZFN 2) Lane 4 and 5 clone 20 (gfpzfn 1and zfn 2)

29. COLONY PCR FOR GFP AND ZFN2 2 kb Out of 10 colonies 2 were positive for gfp

30. IRES BICISTRONIC VECTOR DRIVEN BY CMV PROMOTER IRES GFP ZFN1 ZFN2 ZFN IRES VECTOR ~9.1 kb

31. 1 2 3 4 5 6 E6 PCR LANE 1 LADDER LANE 2 NEGATIVE CONTROL LANE 3 E6 POSITIVE CONTROL LANE 4 HEK TRT WITH ZFNS AT 37 DEG LANE 5 HEK TRT WITH ZFNS AT 30 DEG LANE 6 HEK TRT WITH CAG ZFNS AT 30 DEG 1 2 3 4 5 6 T7E1 ASSAY LANE 1 LADDER LANE 2 E6 POSITIVE CONTROL LANE 3 HEK TRT WITH ZFNS AT 37 DEG LANE 4 HEK TRT WITH CAG ZFNS AT 37 DEG LANE 5 HEK TRT WITH ZFNS AT 30 DEG LANE 6 HEK TRT WITH CAG ZFNS AT 30 DEG

32. Repeated t7E1 assay with reduced concentration of pcr product 1 2 3 4 5 6 7 8 Lane 1 ladder Lane 2 E6 control Lane 6 Hektrt with zfns at 30 deg Lane 7 Hektrt with CAG ZFNs at 30 deg Lane 8 HEK trt with zfns at 37 deg Treatment in HEK

33. 1 2 3 4 5 6 7 T7E1 PRODUCT MORE INTENSE IN LANE 6 T7E1 REPEAT ASSAY Lane 1 ladder Lane 2 E6 control Lane 3 HEK trt with zfns at 37 degrees Lane 4 negative control Lane 5 HEK trt with zfns at 30 degrees Lane 6 HEK trt with cag-zfns at 30 deg Lane 7 HEK trt with cag-zfns at 37 deg

34. 1 2 3 4 5 T7E1 product intense in lane 5 T7E1 ASSAY Lane 1 ladder Lane 2 HEK trt with zfns at 37 degrees Lane 3 negative control Lane 4 HEK trt with zfns at 30 degrees Lane 5 HEK trt with CAG ZFNs at 30 degrees

35. ZFN Activity report based on in-silicoanalysisand a research article

36. SEQUENCING RESULTS • ZFN1 and 2 sequencing was done and it was found to match the ZFN sequences synthesized by sigma. • Following 2 slides are the sequencing results for ZFN 1 & 2

37. ZFN1 SEQUENCING READ

38. ZFN 2 SEQUENCING READ

39. SIGMA’s CERTIFICATE OF ANALYSIS

40. Here is the Certificate of Analysis issued by sigma for E6 ZFN, where the ZFN binding site (in black) and cut site (red) are mentioned From the figure above, the target site has 6 ZFN binding sites on either side of the cut site as indicated by the blue lines ( each zinc finger recognizes a DNA triplet). The cut spacer site indicated in red has 5 nucleotides So the following diagram is the representative image for E6 ZFN with 6 zinc fingers on either side So the following diagram is the representative image for E6 ZFN with 6 zinc fingers on either side

41. THEORETICAL TRANSLATION OF ZFNS TO IDENTIFY THE NUMBER O ZINC FINGERS IN OUR ZINC FINGER PAIR

42. ZFN1 TRANSLATION ZFN2 TRANSLATION

43. Using the motif scan program, it was found that there were totally 12 zinc fingers in the zinc finger plasmid pair that we obtained from sigma Zfn1= 6 fingers Zfn2= 6 fingers

44. ZFN1 MOTIF

45. ZFN2 MOTIF

46. As per the data sheet obtained from Sigma, their Mel1 assay in yeast system showed a good activity of the ZFNs after 6 hours as can be seen in the next image

48. BUT CEL1 assay performed in SiHa cells did not yield any satisfactory results although a faint band was observed after many experiments and standardizations

49. CEL 1 ASSAY There was a very faint cut product which was obtained by Cel 1 assay

50. ZFN ANALYSIS