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1 PRESENT
2 ipscs SafeGuard Fang Yiming He Dawei Zhao Yuchen Chen Haoqi Sun Mengyi
3 Possibility of Regeneration 3
4 Promising Prospect in Medical Application 4
5 ( Stem Cell Technology 5
6 (Keisuke Okita, et al. Nature, 2007) 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 6
7 ips Safeguard 7
8 ips Safeguard Part I: Killer---Suicide gene Part II: Part III: To prevent Unwanted Cells Killer wanted Cells 8
9 Now let me show you our design & results 9
10 Promoter Suicide gene P CMV CANDIDATES for SUICIDE GENE hbax hbax -184 RIP1 RIP3 Apoptin Caspase family Candidates are chosen from genes playing important roles in cell apoptosis pathways Design & Results 10
11 488nm BL HepG2 (DAPI staining) HepG2(BL) HEK293(BL) Killer Suicide genes RIP1 RIP3 and Apoptin successfully induce cell death GFP RIP 1 RIP3 HepG2 Cell Survival Rate (%) ipsc Survival Rate (%) Mock Rip1 Rip3 Apoptin GFP 0 Mock Rip1 Rip3 Apoptin 100μm 100μm 100μm Data is from Flow Cytometry Method(FCM) Apoptin 0h 48h 100μm 100μm 100μm Design & Results 11
12 SELECTIVE KILLING Normally differentiated cells WHAT IS NEEDED? Signal: endogenous and distinguishing molecular markers Pre-transcriptional level Post-transcriptional level : 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 Undifferentiated ips cells & cancer cells Design & Results 12
13 Strategy A : Pre-transcriptional level Signals: transcription factors epigenetic modifications, etc : tissue-specific promoter However Escape of cancer cells: Tissue-specific promoters cannot be universally activated Wanted cells in all types of cancer cells, which may all be differentiated from ips cells. ips cells Unwanted cells Tissue-specific Promoter Suicide gene Design & Results 13
14 Strategy B : Post-transcriptional level Signals: tissue-specific mirna : mirna binding targets on mrna Suicide gene mirna targets mirna mrna Wanted cells mrna degradation Suicide gene mirna targets mrna Unwanted cells Suicide gene expression Design & Results 14
15 A model is found mirna-122 mirna-122 Liver cells Non-liver cells Human liver cells (hepatocyte) Data from Human mirna-122 (endogenous) Design & Results 15
16 PROJECT DECISION Model : Molecular marker: : Human Liver cell (hepatocyte) mirna-122 mirna-122 targets Promoter Suicide gene mirna-122 target Liver cells Liver tissue ips cells Non-liver cells suicide Non-liver cells have included all unwanted cells: undifferentiated ips cells and cancer cells Design & Results 16
17 Promoter GFP mirna-122 target CONSTRUCTION OF SENSOR Completely complementary binding sequence of mirna-122 Natural mirna-122 binding sequence (Partially complementary) DESIGN & RESULTS 17
18 How to test the sensor? Promoter GFP mirna-122 target Low transfection efficiency High transfection efficiency Endogenous mirna-122 Endogenous mirna-122 Liver cells HEK 293T cells DESIGN & RESULTS 18
19 mirna-122 gradients by exogenous expression pmir-122 mirna-122 expressing plasmid HEK 293T cells mirna-122 gradient DESIGN & RESULTS 19
20 mirna-122 Target Responds Accordingly with mir-122 Level pmir-122/ug GFP-target /ug mirna-122 GFP GAPDH p mir-122/ug DESIGN & RESULTS 20
21 DESIGN & RESULTS 21
22 ON ipscells Liver cells Cancer cells OFF : Tet-off system DESIGN & RESULTS 22
23 Leaky Expression of Different TRE OFF ON OFF ing Performance of P TIGHT DESIGN & RESULTS 23
24 Dox has little or no toxicity on m ipscs 100 ipsc survival rate (%) Mock DOX Mock+Dox + DOX DESIGN & RESULTS 24
25 All Parts Assembled DESIGN & RESULTS 25
26 Pef-1α tta tta tta DOX TRE Pmincmv Suicide gene mirna-122 target DESIGN & RESULTS 26
27 X 1 = X 2 = D = = X 1 + D X 1 D k 1 k 2 X 1 D X 2 D k 3 X 2 X 2 k 4 Chemical reactions of gene expression ASSEMBLY WORK 27
28 k 1 X 1 d X 2 dt D = k 1 X 1 D = k 2 X 1 D + k 3 D k 4 X 2 D + X 1 D = D 0 = c 0 X 1 = c 1 y = β α e at + β α α = k 4 β = k 2hc 0 c hc + kc hc 1 1. Solution of ODEs:protein concentration versus time. 2. Dynamics and steady state are determinedby reaction parameters Protein of interest α β Time ASSEMBLY WORK 28
29 P EF-α 4XCUTL P EF-α (weak) 2XCUTL 2XComplete TRE3G P mincmv 2XCUTL P EF-α (Strong) TRE2 P mincmv 4XComplete Tight P mincmv 2XComplete TRE P mincmv P mincmv DOX DOX 0.85 Knockdown Efficiency Knockdown effect of different targets 0.7 Target Type 2*com 2*com+2*CUTL 4*com ASSEMBLY WORK 29
30 P EF-α 4XCUTL P EF-α (weak) y = β α e at + β α 2XCUTL 2XComplete TRE3G P mincmv α = k 4 2XCUTL P EF-α (Strong) Tight TRE2 P mincmv P mincmv 4XComplete β = k 2hc 0 c hc + kc hc 1 2XComplete TRE P mincmv P mincmv ASSEMBLY WORK 30
31 ASSEMBLY WORK 31
32 ipscs Cultivation hipsc on feeder hipsc on matrigel mipsc on feeder mipsc on feeder ASSEMBLY WORK 32
33 Infection of different cell lines mipsc HepG 2 HEK293 Hela U2OS HTC75 HepG2 stable cell line Day 0 Day 2 Day 3 Day 7 ASSEMBLY WORK 33
34 A brief sum up 34
35 Achivements Test every part independently Submit several biobricks and use multiple methods to thoroughly characterized them BBa_K BBa_K BBa_K BBa_K BBa_K BBa_K BBa_K BBa_K BBa_K Set up the foundation for future igem teams to work about ips cells. Summary & Future work 35
36 Future work Test the circuit in vivo; Induce our engineered ipsc into liver cells. Summary & Future work 36
37 Extensions Protect other organs; Extend the device to gene therapy. Summary & Future work 37
38 People's knowledge about ips safeguard Have heard about it 25% have never heard about it 75% Have heard about it have never heard about it Human Practice Summary & Future work 38
39 Instructors Acknowlegement Sponsor Advisor Summary & Future work 39
40 Thank you all!!! Summary & Future work 40
41 Experiments on ipscs hipsc on feeder hipsc on matrigel Design & Results 41
42 Mouse Primary Hepatocytes Hepatocytes HEK pmir ug 1ug Design & Results 42
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DOI: 10.1038/ncb1875 Figure S1 (a) The 79 surgical specimens from NSCLC patients were analysed by immunohistochemistry with an anti-p53 antibody and control serum (data not shown). The normal bronchi served
Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained
Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained with MitoTracker (red), then were immunostained with
SUPPLEMENTARY INFORMATION. Supp. Fig. 1. Autoimmunity. Tolerance APC APC. T cell. T cell. doi: /nature06253 ICOS ICOS TCR CD28 TCR CD28
Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1 Supp. Fig. 2 CD4 + CD44 low CD4
mirna Dr. S Hosseini-Asl
mirna Dr. S Hosseini-Asl 1 2 MicroRNAs (mirnas) are small noncoding RNAs which enhance the cleavage or translational repression of specific mrna with recognition site(s) in the 3 - untranslated region
(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)
Supplementary Figure 1. Functional enrichment analyses of secretomic proteins. (a) Significant biological processes (upper panel) and disease biomarkers (lower panel) 2 involved by hrab37-mediated secretory
Nature Genetics: doi: /ng Supplementary Figure 1
Supplementary Figure 1 MSI2 interactors are associated with the riboproteome and are functionally relevant. (a) Coomassie blue staining of FLAG-MSI2 immunoprecipitated complexes. (b) GO analysis of MSI2-interacting
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein content relative to GAPDH in two independent experiments.
Human induced pluripotent stem cell derived cardiomyocytes are a more relevant model for assessing drug-induced effects on mitochondrial function
Human induced pluripotent stem cell derived cardiomyocytes are a more relevant model for assessing drug-induced effects on mitochondrial function than H9C2 cells 16 January 2013 SLAS2013 Presentation Outline
ACC ELOVL MCAD. CPT1α 1.5 *** 0.5. Reverbα *** *** 0.5. Fasted. Refed
Supplementary Figure A 8 SREBPc 6 5 FASN ELOVL6.5.5.5 ACC.5.5 CLOCK.5.5 CRY.5.5 PPARα.5.5 ACSL CPTα.5.5.5.5 MCAD.5.5 PEPCK.5.5 G6Pase 5.5.5.5 BMAL.5.5 Reverbα.5.5 Reverbβ.5.5 PER.5.5 PER B Fasted Refed
Trim29 gene-targeting strategy. (a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/ ) and homozygous mice ( / ).
Supplementary Figure 1 Trim29 gene-targeting strategy. (a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/ ) and homozygous mice ( / ). (b) Immunoblot analysis of TRIM29 in lung primary
Plasma exposure levels from individual mice 4 hours post IP administration at the
Supplemental Figure Legends Figure S1. Plasma exposure levels of MKC-3946 in mice. Plasma exposure levels from individual mice 4 hours post IP administration at the indicated dose mg/kg. Data represent
Supplementary Figure 1 Cell line TRIB2 status. Supplementary Figure 2 TRIB2 status has no impact on the cell cycle after PI3K inhibition. a. b.
Supplementary Figure 1 Cell line TRIB2 status. TRIB2 protein expression to determine endogenous expression and to determine the effectiveness of each of our TRIB2 knockdown constructs. Supplementary Figure
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DOI: 10.1038/ncb2157 Figure S1 Immobilization of histone pre-mrna to chromatin leads to formation of histone locus body with associated Cajal body. Endogenous histone H2b(e) pre-mrna is processed with
Supplementary Fig. S1. Schematic diagram of minigenome segments.
open reading frame 1565 (segment 5) 47 (-) 3 5 (+) 76 101 125 149 173 197 221 246 287 open reading frame 890 (segment 8) 60 (-) 3 5 (+) 172 Supplementary Fig. S1. Schematic diagram of minigenome segments.
Profiles of gene expression & diagnosis/prognosis of cancer. MCs in Advanced Genetics Ainoa Planas Riverola
Profiles of gene expression & diagnosis/prognosis of cancer MCs in Advanced Genetics Ainoa Planas Riverola Gene expression profiles Gene expression profiling Used in molecular biology, it measures the
Supplementary Figure 1
Supplementary Figure 1 YAP negatively regulates IFN- signaling. (a) Immunoblot analysis of Yap knockdown efficiency with sh-yap (#1 to #4 independent constructs) in Raw264.7 cells. (b) IFN- -Luc and PRDs
SUPPLEMENTARY LEGENDS...
TABLE OF CONTENTS SUPPLEMENTARY LEGENDS... 2 11 MOVIE S1... 2 FIGURE S1 LEGEND... 3 FIGURE S2 LEGEND... 4 FIGURE S3 LEGEND... 5 FIGURE S4 LEGEND... 6 FIGURE S5 LEGEND... 7 FIGURE S6 LEGEND... 8 FIGURE
microrna Presented for: Presented by: Date:
microrna Presented for: Presented by: Date: 2 micrornas Non protein coding, endogenous RNAs of 21-22nt length Evolutionarily conserved Regulate gene expression by binding complementary regions at 3 regions
Soft Agar Assay. For each cell pool, 100,000 cells were resuspended in 0.35% (w/v)
SUPPLEMENTARY MATERIAL AND METHODS Soft Agar Assay. For each cell pool, 100,000 cells were resuspended in 0.35% (w/v) top agar (LONZA, SeaKem LE Agarose cat.5004) and plated onto 0.5% (w/v) basal agar.