SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming Z hao Yuchen L - - PowerPoint PPT Presentation

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SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming Z hao Yuchen L - - PowerPoint PPT Presentation

Sep 09, 2022 287 likes •873 views

SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming Z hao Yuchen L in Kaixuan C hen Haoqi S un Mengyi Never Return Back? 3 A Revolution in View 4 Promising Prospect in Medical Application 5

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SYSU-CHINA@iGEM PRESENT

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iPSCs SafeGuard

Fang Yiming Zhao Yuchen Lin Kaixuan Chen Haoqi Sun Mengyi

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3

Never Return Back?

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4

A Revolution in View

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5

 

Promising Prospect in Medical Application

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Stem Cell Technology

(http://www2.estrellamountain.edu/)

  

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7 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

 

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8  Optimizing viral vectors  Use non-vector methods  Still have risk of carcinogenesis  Induction rate is even lower that can’t be put into use

Trying to Optimize The Methods

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To Choose A Model

(http://www2.estrellamountain.edu/)

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  • （W. Ray Kim et al, Gastroenterology, 2009）

Our Model ---- Liver Cell

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iPS Safeguard

 

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Now let me show you how we design it.

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DESIGN IGN 13

To Liver or Not to Live!

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DESIGN IGN 14

iPSC Safeguard miRNA Target Suicide Gene Tet-off System Lentivirus

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DESIGN IGN 15

Target Suicide Gene Tet-off Lentivirus

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DESIGN IGN 16

Gene Parts  miRNA Target  Suicide Gene  Tet-off System

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DESIGN IGN 17

miRNA-122

Target Suicide Gene Tet-off Lentivirus

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DESIGN IGN 18

Mechanism of miRNA-122

Target Suicide Gene Tet-off Lentivirus

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DESIGN IGN 19

Liver Specific

Target Suicide Gene Tet-off Lentivirus

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DESIGN IGN 20

Promoter Suicide gene miRNA-122 target Target Suicide Gene Tet-off Lentivirus

Suicide Gene

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DESIGN IGN 21

Suicide Gene

 rip1  rip3  Caspase family  hBax  apoptin

Target Suicide Gene Tet-off Lentivirus

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DESIGN IGN 22

Suicide gene miRNA-122 target Promoter

Target Suicide Gene Tet-off Lentivirus

Tet-off System

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DESIGN IGN 23

iPS cells Liver cells Cancer cells

Target Suicide Gene Tet-off Lentivirus

miRNA-122 in Different Cells

miRNA-122

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DESIGN IGN 24

Switch: Tet-off system

Target Suicide Gene Tet-off Lentivirus

ON OFF

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DESIGN IGN

Target Suicide Gene Tet-off Lentivirus

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A Global View

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DESIGN IGN

Target Suicide Gene Tet-off Lentivirus

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DESIGN IGN

Target Suicide Gene Tet-off Lentivirus

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tTA Pef-1α Pmincmv Suicide gene miRNA-122 target TRE DOX tTA tTA

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Now let me show you the results in 4 parts:

Target Suicide Gene Tet-off Lentivirus

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Results ts 29

Strategy

 Screen  Test for the best  Safety& compatibility  Assembly

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Results ts 30

miRNA-122

Searching for the miRNA-122 Target

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Results ts 31

miR-122 Target Responds Accordingly with miRNA-122 Level

GFP GAPDH miR-122

pmiRNA-122/ng 25 50 100 250 GFP-target /ng 750 750 750 750 750

0.00 0.25 0.50 0.75 1.00 25 50 100 250

p miRNA-122/ng

Expression level

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Results ts 32

Corresponding Level of miRNA-122

0.00 3.00 6.00 9.00 12.00 15.00

— — 0.25ug 1ug

HEK293 Hepatocytes

P miRNA-122 400 800 1200 0ug 0.025ug 0.05ug 0.1ug 0.25ug 0.5ug p miRNA-122/ng

0 25 50 100 250 500

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Results ts 33

The Suicide Gene

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Results ts 34

RIP1 and RIP3 successfully induce cell death

HEK293 (BL) HepG2 (BL)

GFP RIP 1

HepG2 (DAPI)

RIP3

100μm 100μm 100μm 100μm 100μm 100μm
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Results ts 35

ApoptinSuccessfully Induces Cell Death

100μm 100μm

0h 48h Bright Light

Apoptin-GFP induced cell death

488nm

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Results ts 36

Profile of the Suicide Genes

25 50 75 100 Mock Rip1 Rip3 Apoptin GFP

HepG2 Cell Survival Rate (%)

25 50 75 Mock Rip1 Rip3 Apoptin

iPSC Survival Rate (%)

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Results ts 37

Tet-off System

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Results ts 38

Leaky Expression of Different TRE

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Results ts 39

Switching Performance of PTIGHT

ON OFF

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Results ts 40

Test in Mouse iPSCs

20 40 60 80

Mock TRE RE3G - rip ip1 TRE RE3G - rip ip3 TRE RE3G - GFP

iPSC suvival rate (%)

25 50 75 100

Mock Mock+Dox

iPSC survival rate (%)

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Results ts 41

Target

Everybody Comes Together!

Suicide Gene Tet-off Lentivirus

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Results ts 42

Lentivirus Packaging

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Results ts 43

Functional tests in different cell lines

HepG 2 HEK293 Hela U2OS HTC75

Day 0 Day 2 Day 3 Day 7 HepG2 stable cell line

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Now let me show you the Tuningand Modeling works

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Modeling ing 45

Fine-tune the variation range of knockdown efficiency

miRNA-122

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Modeling ing 46 Knockdown effect of different targets

0.7 0.75 0.8 0.85

2*com 2*com+2*CUTL 4*com

Knockdown Efficiency

Target Type

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Modeling ing 47

Modulation of MiR122-Target device

Interpolation Method

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Modeling ing 48 d 1=- k1 1 dt d 2 =k1 1 k2 2 dt d 3 =k2 2 dt X X X X X X X 

d 1=- k1 1 dt d 2 =n1k1 1 k2 2 dt d 3 =n2k2 2 dt X X X X X X X 

X1 ,X2 ,X3 k1 ,k2 ,k3

Multiple-Compartment Model

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Modeling ing 49

Sum up

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Now let me show you our future work

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Futu ture e Work 51

Achivements

 Sucide genes  miRNA-122  Tet-off

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Futu ture e Work 52

To be Continue……

 Apoptosis-related gene  Do the experiments in stable cell lines  Complete circuit in human iPS cells

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Futu ture e Work 53

Extension

(Thomas M. Schmitt et al,Hum Gene Ther.2009)

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Futu ture e Work 54

Human Practice

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Futu ture e Work 55

Acknowlegement

Instructors Advisor Colleage Sponsor

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Futu ture e Work 56

Thank you all!!!

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Results ts 57

Experiments on iPSCs

hiPSC on feeder hiPSC on matrigel

66.7 6.42 8.39 9.43 10 20 30 40 50 60 70 80

Mock rip1 rip3 apoptin

iPSC Survival Rate(%)

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Results ts 58

Mouse Primary Hepatocytes

11.29 1.00 7.02 8.22 2 4 6 8 10 12 14 — — 0.25ug 1ug pMiR-122

HEK293 Hepatocytes