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SYSU-CHINA@iGEM PRESENT
iPSCs
SafeGuard
Fang Yiming He Dawei Zhao Yuchen Chen Haoqi Sun Mengyi
3
Possibility of
Regeneration
4
Promising Prospect in Medical Application
5
Stem Cell Technology
(http://www2.estrellamountain.edu/)
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)
7
iPS Safeguard
8
Part I: Killer---Suicide gene
To prevent
Sensor
Part II: Sensor
iPS Safeguard
Switch
Part III: Switch
Unwanted Cells
wanted Cells
Killer
9
Now let me show you our design & results
Killer Sensor Switch
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
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
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
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
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
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
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
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
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
19DESIGN & RESULTS
miRNA-122 gradients by exogenous expression
pMiR-122
HEK 293T
cells
miRNA-122 gradient
miRNA-122
expressing
plasmid
SensorKiller Switch
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
21DESIGN & RESULTS
22DESIGN & RESULTS
Switch: Tet-off system
ON OFF
iPScells Liver cells Cancer cells
SensorKiller Switch
23DESIGN & RESULTS
Leaky Expression of Different TRE
SensorKiller Switch
Switching Performance of PTIGHT
ONOFF OFF
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
DESIGN & RESULTS 25
SensorKiller Switch
All Parts Assembled
DESIGN & RESULTS 26
SensorKiller Switch
tTAPef-1α
Pmincmv Suicide gene miRNA-122 targetTRE
DOXtTA
tTA
ASSEMBLY WORK 27
SensorKiller Switch
Chemical reactions of gene expression
𝑿𝟏 +𝑫𝒌𝟏
𝑿𝟏𝑫
𝑿𝟏𝑫𝒌𝟐
𝑿𝟐
𝑫𝒌𝟑
𝑿𝟐
𝑿𝟐𝒌𝟒
∅
𝑿𝟏 = 𝑿𝟐 =𝑫 = ∅ =
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
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
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
ASSEMBLY WORK 31
SensorKiller Switch
ASSEMBLY WORK 32
SensorKiller Switch
iPSCs Cultivation
hiPSC on feeder hiPSC on matrigel
miPSC on feeder miPSC on feeder
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
34
A brief sum up
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
Summary & Future work 36
Future work
Test the circuit in vivo;
Induce our engineered iPSc into liver cells.
Summary & Future work 37
Extensions
Protect other organs;
Extend the device to gene therapy.
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
Summary & Future work 39
Acknowlegement Instructors
Advisor
Sponsor
Summary & Future work 40
Thank you all!!!
Design & Results 41
Experiments on iPSCs
hiPSC on feeder hiPSC on matrigel
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
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