Post on 27-Apr-2020
SYSU-CHINA@iGEM PRESENT
iPSCs
SafeGuard
Fang Yiming Zhao Yuchen Lin Kaixuan Chen Haoqi Sun Mengyi
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Never Return Back?
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A Revolution in View
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Promising Prospect in Medical Application
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Stem Cell Technology
(http://www2.estrellamountain.edu/)
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In Yamanaka’s experiment in 2009, the tumor formation rate is 30%among 100 mice transplanted with iPS cells, much higher thannorman ES cells. That’s due to: Reactivation of transcription factor c-Myc, also an oncogene Wrong insertion of viral vectors
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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.
DESIGN 13
To Liver or Not to Live!
DESIGN 14
iPSC Safeguard
miRNA TargetSuicide GeneTet-off System
Lentivirus
DESIGN 15
Target Suicide Gene Tet-offLentivirus
DESIGN 16
Gene Parts
miRNA Target Suicide Gene Tet-off System
DESIGN 17
miRNA-122
Target Suicide Gene Tet-offLentivirus
DESIGN 18
Mechanism of miRNA-122
Target Suicide Gene Tet-offLentivirus
DESIGN 19
Liver Specific
Target Suicide Gene Tet-offLentivirus
DESIGN 20
Promoter
Suicide gene miRNA-122 target
Target Suicide Gene Tet-offLentivirus
Suicide Gene
DESIGN 21
Suicide Gene
rip1 rip3 Caspase family hBax apoptin
Target Suicide Gene Tet-offLentivirus
DESIGN 22
Suicide gene
miRNA-122 target
Promoter
Target Suicide Gene Tet-offLentivirus
Tet-off System
DESIGN 23
iPS cells
Liver cells
Cancer cells
Target Suicide Gene Tet-offLentivirus
miRNA-122 in Different Cells
miRNA-122
DESIGN 24
Switch: Tet-off system
Target Suicide Gene Tet-offLentivirus
ON OFF
DESIGN
Target Suicide Gene Tet-offLentivirus
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A Global View
DESIGN
Target Suicide Gene Tet-offLentivirus
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DESIGN
Target Suicide Gene Tet-offLentivirus
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tTAPef-1α
Pmincmv Suicide gene miRNA-122 targetTRE
DOXtTA
tTA
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Now let me show you the results in 4 parts:
Target Suicide Gene Tet-off Lentivirus
Results 29
Strategy
Screen Test for the best Safety& compatibility Assembly
Results 30
miRNA-122
Searching for the miRNA-122 Target
Results 31
miR-122 Target Responds Accordingly with miRNA-122 Level
GFP
GAPDH
miR-122
pmiRNA-122/ng 0 25 50 100 250
GFP-target /ng 750 750 750 750 750
0.00
0.25
0.50
0.75
1.00
0 25 50 100 250
p miRNA-122/ng
Expression level
Results 32
Corresponding Level of miRNA-122
0.00
3.00
6.00
9.00
12.00
15.00
— — 0.25ug 1ug
HEK293Hepatocytes
P miRNA-122
0
400
800
1200
0ug 0.025ug 0.05ug 0.1ug 0.25ug 0.5ugp miRNA-122/ng
0 25 50 100 250 500
Results 33
The Suicide Gene
Results 34
RIP1 and RIP3 successfully induce cell death
HEK293(BL)
HepG2(BL)
GFP RIP 1
HepG2(DAPI)
RIP3
100μm100μm 100μm
100μm 100μm 100μm
Results 35
Apoptin Successfully Induces Cell Death
100μm 100μm
0h 48h
Bright Light
Apoptin-GFP induced cell death
488nm
Results 36
Profile of the Suicide Genes
0
25
50
75
100
Mock Rip1 Rip3 Apoptin GFP
HepG2 Cell Survival Rate (%)
0
25
50
75
Mock Rip1 Rip3 Apoptin
iPSC Survival Rate (%)
Results 37
Tet-off System
Results 38
Leaky Expression of Different TRE
Results 39
Switching Performance of PTIGHT
ON OFF
Results 40
Test in Mouse iPSCs
0
20
40
60
80
Mock TRE3G - rip1 TRE3G - rip3 TRE3G - GFP
iPSC suvival rate (%)
0
25
50
75
100
Mock Mock+Dox
iPSC survival rate (%)
Results 41
Target
Everybody Comes Together!
Suicide Gene Tet-off Lentivirus
Results 42
Lentivirus Packaging
Results 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 Modelingworks
Modeling 45
Fine-tune the variation range of knockdown efficiency
miRNA-122
Modeling 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
Modeling 47
Modulation of MiR122-Target device
Interpolation Method
Modeling 48
d 1=- k1 1
dt
d 2=k1 1 k2 2
dt
d 3=k2 2
dt
XX
XX X
XX
d 1=- k1 1
dt
d 2=n1k1 1 k2 2
dt
d 3=n2k2 2
dt
XX
XX X
XX
X1 ,X2 , X3 k1 ,k2 , k3
Multiple-Compartment Model
Modeling 49
Sum up
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Now let me show you our future work
Future Work 51
Achivements
Sucide genes miRNA-122 Tet-off
Future Work 52
To be Continue……
Apoptosis-related gene
Do the experiments in stable cell lines
Complete circuit in human iPS cells
Future Work 53
Extension
(Thomas M. Schmitt et al,Hum Gene Ther.2009)
Future Work 54
Human Practice
Future Work 55
Acknowlegement
Instructors
Advisor
Colleage Sponsor
Future Work 56
Thank you all!!!
Results 57
Experiments on iPSCs
hiPSC on feeder hiPSC on matrigel
66.7
6.42 8.39 9.43
0
10
20
30
40
50
60
70
80
Mock rip1 rip3 apoptin
iPSC Survival Rate(%)
Results 58
Mouse Primary Hepatocytes
11.29
1.00
7.02
8.22
0
2
4
6
8
10
12
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— — 0.25ug 1ug
pMiR-122
HEK293Hepatocytes