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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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6
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 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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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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9
To Choose A Model
(http://www2.estrellamountain.edu/)
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10
• (W. Ray Kim et al, Gastroenterology, 2009)
Our Model ---- Liver Cell
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11
iPS Safeguard
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12
Now let me show you how we design it.
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DESIGN 13
To Liver or Not to Live!
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DESIGN 14
iPSC Safeguard
miRNA TargetSuicide GeneTet-off System
Lentivirus
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DESIGN 15
Target Suicide Gene Tet-offLentivirus
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DESIGN 16
Gene Parts
miRNA Target Suicide Gene Tet-off System
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DESIGN 17
miRNA-122
Target Suicide Gene Tet-offLentivirus
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DESIGN 18
Mechanism of miRNA-122
Target Suicide Gene Tet-offLentivirus
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DESIGN 19
Liver Specific
Target Suicide Gene Tet-offLentivirus
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DESIGN 20
Promoter
Suicide gene miRNA-122 target
Target Suicide Gene Tet-offLentivirus
Suicide Gene
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DESIGN 21
Suicide Gene
rip1 rip3 Caspase family hBax apoptin
Target Suicide Gene Tet-offLentivirus
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DESIGN 22
Suicide gene
miRNA-122 target
Promoter
Target Suicide Gene Tet-offLentivirus
Tet-off System
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DESIGN 23
iPS cells
Liver cells
Cancer cells
Target Suicide Gene Tet-offLentivirus
miRNA-122 in Different Cells
miRNA-122
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DESIGN 24
Switch: Tet-off system
Target Suicide Gene Tet-offLentivirus
ON OFF
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DESIGN
Target Suicide Gene Tet-offLentivirus
25
A Global View
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DESIGN
Target Suicide Gene Tet-offLentivirus
26
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DESIGN
Target Suicide Gene Tet-offLentivirus
27
tTAPef-1α
Pmincmv Suicide gene miRNA-122 targetTRE
DOXtTA
tTA
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28
Now let me show you the results in 4 parts:
Target Suicide Gene Tet-off Lentivirus
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Results 29
Strategy
Screen Test for the best Safety& compatibility Assembly
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Results 30
miRNA-122
Searching for the miRNA-122 Target
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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
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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
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Results 33
The Suicide Gene
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Results 34
RIP1 and RIP3 successfully induce cell death
HEK293(BL)
HepG2(BL)
GFP RIP 1
HepG2(DAPI)
RIP3
100μm100μm 100μm
100μm 100μm 100μm
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Results 35
Apoptin Successfully Induces Cell Death
100μm 100μm
0h 48h
Bright Light
Apoptin-GFP induced cell death
488nm
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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 (%)
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Results 37
Tet-off System
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Results 38
Leaky Expression of Different TRE
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Results 39
Switching Performance of PTIGHT
ON OFF
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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 (%)
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Results 41
Target
Everybody Comes Together!
Suicide Gene Tet-off Lentivirus
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Results 42
Lentivirus Packaging
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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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44
Now let me show you the Tuningand Modelingworks
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Modeling 45
Fine-tune the variation range of knockdown efficiency
miRNA-122
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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
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Modeling 47
Modulation of MiR122-Target device
Interpolation Method
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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
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Modeling 49
Sum up
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50
Now let me show you our future work
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Future Work 51
Achivements
Sucide genes miRNA-122 Tet-off
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Future 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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Future Work 53
Extension
(Thomas M. Schmitt et al,Hum Gene Ther.2009)
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Future Work 54
Human Practice
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Future Work 55
Acknowlegement
Instructors
Advisor
Colleage Sponsor
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Future Work 56
Thank you all!!!
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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(%)
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Results 58
Mouse Primary Hepatocytes
11.29
1.00
7.02
8.22
0
2
4
6
8
10
12
14
— — 0.25ug 1ug
pMiR-122
HEK293Hepatocytes