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SYSU-CHINA@iGEM PRESENT
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iPSCs
SafeGuard
Fang Yiming He Dawei Zhao Yuchen Chen Haoqi Sun Mengyi
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3
Possibility of
Regeneration
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4
Promising Prospect in Medical Application
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5
Stem Cell Technology
(http://www2.estrellamountain.edu/)
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6
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
(Keisuke Okita, et al. Nature, 2007)
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7
iPS Safeguard
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8
Part I: Killer---Suicide gene
To prevent
Sensor
Part II: Sensor
iPS Safeguard
Switch
Part III: Switch
Unwanted Cells
wanted Cells
Killer
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9
Now let me show you our design & results
Killer Sensor Switch
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Design & Results 10
Promoter
PCMV
Suicide gene
SensorKiller Switch
hBax hBax -184 RIP1 RIP3 Apoptin Caspase family
Candidates are chosen from genes playing important roles in cell apoptosis pathways
CANDIDATES
for SUICIDE GENE
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Design & Results 11
Suicide genes RIP1 RIP3and Apoptinsuccessfully induce cell death
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 (%)
Data is from Flow Cytometry Method(FCM)
GFP RIP 1 RIP3
HE
K2
93
(BL
)H
ep
G2
(BL
)H
ep
G2
(DA
PI
sta
inin
g)
100μm100μm 100μm
100μm 100μm 100μm
0h 48h
BL
48
8n
m
Apoptin
SensorKiller Switch
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Design & Results 12
Normally differentiated cells
SELECTIVE KILLING
Undifferentiated ips cells
& cancer cells
WHAT IS NEEDED?
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 represent the normally differentiated cells in our project
SensorKiller Switch
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Design & Results 13
Strategy A : Pre-transcriptional level
Signals: transcription factors epigenetic modifications, etc
Sensor:tissue-specific promoter
However…
Escape of cancer cells:Tissue-specific promoters cannot be universally activated in all types of cancer cells, which may all be differentiated from iPS cells.
Wanted cells
Unwanted
cells
iPS
cells
Suicide gene
Tissue-specific
Promoter
SensorKiller Switch
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Design & Results 14
Strategy B : Post-transcriptional level Signals: tissue-specific miRNA Sensor: miRNA binding targets on mRNA
Suicide gene miRNA targetsmRNA
miRNA
Suicide gene miRNA targetsmRNA
Wanted cells
Unwanted cells
mRNA
degradation
Suicide gene
expression
SensorKiller Switch
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Design & Results 15
A model is found
Human miRNA-122 (endogenous)
Human liver cells
(hepatocyte)
SensorKiller Switch
miRNA-122 miRNA-122
Liver cells Non-liver cells
Data from www.microrna.org
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Design & Results 16
PROJECT DECISION
PromoterSuicide gene miRNA-122 target
Liver cells Liver tissue
iPS cells Non-liver cells have included all
unwanted cells: undifferentiated
iPS cells and cancer cellsNon-liver cells suicide
Model : Human Liver cell (hepatocyte)
Molecular marker: miRNA-122
Sensor: miRNA-122 targets
SensorKiller Switch
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17DESIGN & RESULTS
Promoter
GFP miRNA-122 target
Completely complementary
binding sequence of
miRNA-122
Natural miRNA-122 binding
sequence
(Partially complementary)
CONSTRUCTION OF SENSOR
SensorKiller Switch
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18DESIGN & RESULTS
How to test the sensor?Promoter
GFP miRNA-122 target
Low transfection
efficiency
High transfection
efficiency
Liver cells HEK 293T cells
Endogenous
miRNA-122Endogenous
miRNA-122
SensorKiller Switch
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19DESIGN & RESULTS
miRNA-122 gradients by exogenous expression
pMiR-122
HEK 293T
cells
miRNA-122 gradient
miRNA-122
expressing
plasmid
SensorKiller Switch
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20DESIGN & RESULTS
miRNA-122 Target Responds Accordingly with miR-122 Level
0 0.025 0.05 0.1 0.25
GFP
GAPDH
miRNA-122
pmiR-122/ug
p miR-122/ug
GFP-target /ug 0.75 0.75 0.75 0.75 0.75
SensorKiller Switch
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21DESIGN & RESULTS
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22DESIGN & RESULTS
Switch: Tet-off system
ON OFF
iPScells Liver cells Cancer cells
SensorKiller Switch
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23DESIGN & RESULTS
Leaky Expression of Different TRE
SensorKiller Switch
Switching Performance of PTIGHT
ONOFF OFF
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24DESIGN & RESULTS
0
25
50
75
100
Mock Mock+Dox
iPSC survival rate (%)
+ DOX- DOX
Dox has little or no toxicity on m iPSCs
SensorKiller Switch
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DESIGN & RESULTS 25
SensorKiller Switch
All Parts Assembled
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DESIGN & RESULTS 26
SensorKiller Switch
tTAPef-1α
Pmincmv Suicide gene miRNA-122 targetTRE
DOXtTA
tTA
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ASSEMBLY WORK 27
SensorKiller Switch
Chemical reactions of gene expression
𝑿𝟏 +𝑫𝒌𝟏
𝑿𝟏𝑫
𝑿𝟏𝑫𝒌𝟐
𝑿𝟐
𝑫𝒌𝟑
𝑿𝟐
𝑿𝟐𝒌𝟒
∅
𝑿𝟏 = 𝑿𝟐 =𝑫 = ∅ =
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ASSEMBLY WORK 28
SensorKiller Switch
𝒌𝟏 𝑿𝟏 𝑫 = 𝒌−𝟏𝑿𝟏𝑫
𝒅 𝑿𝟐𝒅𝒕
= 𝒌𝟐 𝑿𝟏𝑫 + 𝒌𝟑 𝑫 − 𝒌𝟒 𝑿𝟐
𝑫 + 𝑿𝟏𝑫 = 𝑫𝟎 = 𝒄𝟎
𝑿𝟏 = 𝒄𝟏
𝒚 = −𝜷
𝜶𝒆−𝒂𝒕 +
𝜷
𝜶
𝞪 = 𝒌𝟒
𝞫 = −𝒌𝟐𝒉𝒄𝟎𝒄𝟏𝟏 + 𝒉𝒄
+𝒌𝒄𝟎
𝟏 + 𝒉𝒄𝟏
⟹
1. SolutionofODEs:proteinconcentra-tionversustime.
2. Dynamicsandsteadystatearedeter-minedbyreaction parameters
𝞪
𝞫
Protein of interest
Time
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ASSEMBLY WORK 29
SensorKiller Switch
2XComplete
4XComplete
2XCUTL
2XCUTL 2XComplete
PEF-𝞪
PminCMVTRE PminCMV
Knockdown effect of different targets0.7
0.75
0.8
0.85
2*com 2*com+2*CUTL 4*com
Knockdown Efficiency
Target Type
PEF-𝞪(weak)
PminCMVTight
PminCMVTRE2
PminCMVTRE3G
PEF-𝞪(Strong)
4XCUTL
DOX DOX
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ASSEMBLY WORK 30
SensorKiller Switch
2XComplete
4XComplete
2XCUTL
2XCUTL 2XComplete
PEF-𝞪
PminCMVTRE PminCMV
PEF-𝞪(weak)
PminCMVTight
PminCMVTRE2
PminCMVTRE3G
PEF-𝞪(Strong)
4XCUTL
𝒚 = −𝜷
𝜶𝒆−𝒂𝒕 +
𝜷
𝜶
𝞪 = 𝒌𝟒
𝞫 = −𝒌𝟐𝒉𝒄𝟎𝒄𝟏𝟏 + 𝒉𝒄
+𝒌𝒄𝟎
𝟏 + 𝒉𝒄𝟏
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ASSEMBLY WORK 31
SensorKiller Switch
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ASSEMBLY WORK 32
SensorKiller Switch
iPSCs Cultivation
hiPSC on feeder hiPSC on matrigel
miPSC on feeder miPSC on feeder
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ASSEMBLY WORK 33
SensorKiller Switch
Day 0 Day 2 Day 3 Day 7
HepG2 stable cell line
miPSC
HepG 2
HEK293
Hela
U2OS
HTC75
Infection of different cell lines
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34
A brief sum up
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Summary & Future work 35
Achivements
Test every part independently
Set up the foundation for future iGEM teams
to work about iPS cells.
BBa_K1061001 BBa_K1061002 BBa_K1061003 BBa_K1061006
Submit several biobricks and use multiple
methods to thoroughly characterized them BBa_K1061011 BBa_K1061012 BBa_K1061012 BBa_K1061014
BBa_K1061021
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Summary & Future work 36
Future work
Test the circuit in vivo;
Induce our engineered iPSc into liver cells.
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Summary & Future work 37
Extensions
Protect other organs;
Extend the device to gene therapy.
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Summary & Future work 38
Human Practice
Have heard about it
25%
have never heard about it
75%
People's knowledge about iPS safeguard
Have heard about it have never heard about it
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Summary & Future work 39
Acknowlegement Instructors
Advisor
Sponsor
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Summary & Future work 40
Thank you all!!!
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Design & Results 41
Experiments on iPSCs
hiPSC on feeder hiPSC on matrigel
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Design & Results 42
Mouse Primary Hepatocytes
11.29
1.00
7.02
8.22
0
2
4
6
8
10
12
14
— — 0.25ug 1ugpMiR-122
HEK293Hepatocytes