SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming H e Dawei Z - PowerPoint PPT Presentation

SYSU-CHINA@iGEM PRESENT

iPSCs SafeGuard F ang Yiming H e Dawei Z hao Yuchen C hen Haoqi S un Mengyi

Possibility of Regeneration  3

Promising Prospect in Medical Application   4

  (http://www2.estrellamountain.edu/) Stem Cell Technology 5

   (Keisuke Okita, et al. Nature, 2007) In Yamanaka’s experiment in 2009, the tumor formation rate is 30% among 100 mice transplanted with iPS cells, much higher than norman ES cells. That’s due to:  Reactivation of transcription factor c-Myc, also an oncogene  Wrong insertion of viral vectors 6

iPS Safeguard  7

iPS Safeguard  Part I: Killer---Suicide gene  Part II: Sensor  Part III: Switch To prevent Unwanted Cells Switch Killer Sensor wanted 8 Cells

Sensor Switch Killer Now let me show you our design & results 9

Killer Sensor Switch Promoter Suicide gene P CMV CANDIDATES for SUICIDE GENE hBax   hBax -184  RIP1  RIP3 Apoptin   Caspase family Candidates are chosen from genes playing important roles in cell apoptosis pathways Design ign & Results ts 10

Killer Sensor Switch Suicide genes RIP1 RIP3and Apoptinsuccessfully induce cell death GFP RIP 1 RIP3 HepG2 Cell Survival Rate (%) iPSC Survival Rate (%) 100 75 HEK293(BL) 75 50 50 25 25 100 μ m 100 μ m 100 μ m 0 0 Mock Rip1 Rip3 Apoptin GFP Mock Rip1 Rip3 Apoptin Data is from Flow Cytometry Method(FCM) HepG2(BL) Apoptin 0h 48h 100 μ m 100 μ m 100 μ m (DAPI staining) BL HepG2 488nm Design ign & Results ts 11

Killer Sensor Switch WHAT IS NEEDED? SELECTIVE KILLING  Signal: endogenous and distinguishing molecular markers  Pre-transcriptional level  Post-transcriptional level  Sensor: to sense the signal and determine the expression of suicide gene  A model: one type of somatic cell which Normally differentiated cells Undifferentiated ips cells represent the normally differentiated & cancer cells cells in our project Design ign & Results ts 12

Killer Sensor Switch Strategy A : Pre-transcriptional level  Signals : transcription factors epigenetic modifications, etc  Sensor: tissue-specific promoter However… Tissue-specific  Escape of cancer cells: Promoter Tissue-specific promoters cannot be universally activated Wanted cells in all types of cancer cells, which may all be differentiated Suicide gene from iPS cells. iPS cells Unwanted cells Design ign & Results ts 13

Killer Sensor Switch Strategy B : Post-transcriptional level  Signals: tissue-specific miRNA  Sensor: miRNA binding targets on mRNA miRNA mRNA Suicide gene miRNA targets mRNA Wanted cells degradation mRNA Suicide gene miRNA targets Suicide gene expression Unwanted cells Design ign & Results ts 14

Killer Sensor Switch A model is found miRNA-122 miRNA-122 Liver cells Non-liver cells Human liver cells (hepatocyte) Data from www.microrna.org Human miRNA-122 (endogenous) Design ign & Results ts 15

Killer Sensor Switch PROJECT DECISION Model : Human Liver cell (hepatocyte) Molecular marker: miRNA-122 Sensor: miRNA-122 targets Promoter Suicide gene miRNA-122 target Liver cells Liver tissue Non-liver cells have included all iPS cells unwanted cells: undifferentiated iPS cells and cancer cells Non-liver cells suicide Design ign & Results ts 16

Killer Sensor Switch Promoter GFP miRNA-122 target CONSTRUCTION OF SENSOR Completely complementary binding sequence of miRNA-122 Natural miRNA-122 binding sequence (Partially complementary) DESIGN IGN & RESUL ULTS TS 17

Killer Sensor Switch How to test the sensor? Promoter GFP miRNA-122 target Low transfection High transfection efficiency efficiency Endogenous Endogenous miRNA-122 miRNA-122 Liver cells HEK 293T cells DESIGN IGN & RESUL ULTS TS 18

Killer Sensor Switch miRNA-122 gradients by exogenous expression pMiR-122 miRNA-122 expressing plasmid HEK 293T cells miRNA-122 gradient DESIGN IGN & RESUL ULTS TS 19

Killer Sensor Switch miRNA-122 Target Responds Accordingly with miR-122 Level 0.025 p miR-122/ug 0.05 0.25 0.1 0 GFP-target /ug 0.75 0.75 0.75 0.75 0.75 miRNA-122 GFP GAPDH p miR-122/ug DESIGN IGN & RESUL ULTS TS 20

DESIGN IGN & RESUL ULTS TS 21

Killer Sensor Switch iPScells Liver cells Cancer cells ON OFF Switch: Tet-off system DESIGN IGN & RESUL ULTS TS 22

Killer Sensor Switch Leaky Expression of Different TRE ON OFF OFF Switching Performance of P TIGHT DESIGN IGN & RESUL ULTS TS 23

Killer Sensor Switch  Doxhas little or no toxicity on m iPSCs iPSC survival rate (%) 100 75 50 25 0 Mock Mock+Dox - DOX DOX + DOX + DOX DESIGN IGN & RESUL ULTS TS 24

Killer Sensor Switch All Parts Assembled DESIGN IGN & RESUL ULTS TS 25

Killer Sensor Switch P ef-1 α tTA tTA DOX tTA miRNA-122 target P mincm v Suicide gene TRE DESIGN IGN & RESUL ULTS TS 26

Killer Sensor Switch 𝒀 𝟐 = 𝒀 𝟑 = ∅ = 𝑬 = 𝒍 𝟐 𝒀 𝟐 + 𝑬 𝒀 𝟐 𝑬 𝒍 𝟑 𝒀 𝟐 𝑬 𝒀 𝟑 𝒍 𝟒 𝑬 𝒀 𝟑 𝒍 𝟓 𝒀 𝟑 ∅ Chemical reactions of gene expression ASSEM EMBLY BLY WORK 27

Killer Sensor Switch 𝒍 𝟐 𝒀 𝟐 𝑬 = 𝒍 −𝟐 𝒀 𝟐 𝑬 𝒛 = − 𝜸 𝜷 𝒇 −𝒃𝒖 + 𝜸 𝜷 𝒆 𝒀 𝟑 = 𝒍 𝟑 𝒀 𝟐 𝑬 + 𝒍 𝟒 𝑬 − 𝒍 𝟓 𝒀 𝟑 𝒆𝒖 𝞫 = 𝒍 𝟓 ⟹ 𝑬 + 𝒀 𝟐 𝑬 = 𝑬 𝟏 = 𝒅 𝟏 𝞬 = − 𝒍 𝟑 𝒊𝒅 𝟏 𝒅 𝟐 𝒍𝒅 𝟏 𝟐 + 𝒊𝒅 + 𝒀 𝟐 = 𝒅 𝟐 𝟐 + 𝒊𝒅 𝟐 1. SolutionofODEs:proteinconcentra- Protein of interest tionversustime. 2. Dynamicsandsteadystatearedeter- minedbyreactionparameters 𝞫 Time 𝞬 ASSEM EMBLY BLY WORK 28

Killer Sensor Switch 4XCUTL P EF- 𝞫 2XCUTL 2XComplete P EF- 𝞫 (weak) P minCMV TRE3G 2XCUTL P EF- 𝞫 (Strong) 4XComplete P minCMV TRE2 2XComplete P minCMV Tight P minCMV TRE P minCMV DOX DOX Knockdown Efficiency 0.85 0.8 0.75 0.7 Knockdown effect of different targets 2*com 2*com+2 +2*C *CUTL 4*com Target Type ASSEM EMBLY BLY WORK 29

Killer Sensor Switch 4XCUTL P EF- 𝞫 𝒛 = − 𝜸 𝜷 𝒇 −𝒃𝒖 + 𝜸 2XCUTL 2XComplete 𝜷 P EF- 𝞫 (weak) 𝞫 = 𝒍 𝟓 P minCMV TRE3G 2XCUTL P EF- 𝞫 (Strong) 𝞬 = − 𝒍 𝟑 𝒊𝒅 𝟏 𝒅 𝟐 𝒍𝒅 𝟏 4XComplete P minCMV TRE2 𝟐 + 𝒊𝒅 + 𝟐 + 𝒊𝒅 𝟐 2XComplete P minCMV Tight P minCMV TRE P minCMV ASSEM EMBLY BLY WORK 30

Killer Sensor Switch ASSEM EMBLY BLY WORK 31

Killer Sensor Switch iPSCs Cultivation hiPSC on feeder hiPSC on matrigel miPSC on feeder miPSC on feeder ASSEM EMBLY BLY WORK 32

Killer Sensor Switch Infection of different cell lines HepG2 stable cell line  miPSC  HepG2  HEK293  Hela  U2OS Day 0 Day 2 Day 3 Day 7  HTC75 ASSEM EMBLY BLY WORK 33

A brief sum up 34

Achivements  Test every part independently  S ubmit several biobricks and use multiple methods to thoroughly characterized them  BBa_K1061001  BBa_K1061011  BBa_K1061021  BBa_K1061002  BBa_K1061012  BBa_K1061003  BBa_K1061012  BBa_K1061006  BBa_K1061014  S et up the foundation for future iGEM teams to work about iPS cells. Summa mmary y & Futu ture e work 35

Future work  Test the circuit in vivo;  Induce our engineered iPSc into liver cells. Summa mmary y & Futu ture e work 36

Extensions  Protect other organs;  Extend the device to gene therapy. Summa mmary y & Futu ture e work 37

People's knowledge about iPS safeguard Have heard about it 25% have never heard about it 75% Have heard about it have never heard about it Human Practice Summa mmary y & Futu ture e work 38

Acknowlegement  Instructors  Sponsor  Advisor Summa mmary y & Futu ture e work 39

Thank you all!!! Summa mmary y & Futu ture e work 40

Experiments on iPSCs hiPSC on feeder hiPSC on matrigel Design ign & Results ts 41

Mouse Primary Hepatocytes 14 Hepatocytes HEK293 12 11.29 10 8.22 8 7.02 6 4 2 1.00 0 pMiR-122 — — 0.25ug 1ug Design ign & Results ts 42