Accelerating discoveries through innovations
PRODUCT BROCHURE Vector Technologies • MicroRNA • Exosomes • Stem Cells • Libraries
Spend less time making tools, and more time making discoveries
LENTIVECTOR SYSTEMS
Efficient Delivery & Stable Expression The human (HIV) and feline (FIV) Lentiviral Vector Systems provide exceptional tools to express any sequence in virtually all mammalian cells and in vivo models. The lentiviral system consists of three main components: (1) the lentiviral expression plasmids (also known as "transfer vector") (2) the lentiviral packaging plasmid mix PROMOTER OPTIONS MARKER OPTIONS (3) a producer cell line for packaging CMV
Lentivector formats • Stably Overexpress cDNAs and MicroRNAs Choose from single or double promoter formats featuring CMV, EF1, PGK, UbC or MSCV promoter options with fluorescent proteins and/or antibiotic markers. • Permanent Gene Knockdown with shRNA Overexpression These vectors use a robust H1 promoter to drive shRNA expression, with different maker combinations including the popular pGreenPuro dual marker shRNA vector.
GFP
EF1 alpha
RFP
MSCV
PURO
PGK
ZEO
UBC
HYGRO
Tissue-specific
NEO
Cumate Switch Inducible
THYMIDINE KINASE (TK)
VECTOR FORMATS
LUCIFERASE (LUC)
cDNA
GFP-T2A-PURO
shRNA
RFP-T2A-PURO
microRNA or anti-microRNA
GFP-T2A-LUC
Promoter Reporters
RFP-T2A-LUC
Transcription Response Reporters GFP-T2A-Zeo
Inducible Cumate Switch Vectors
Cumate Switch
Other Systems
- Cumate
- Inducer
+ Cumate
+ Inducer
The Cumate Switch SparQ™ system delivers extremely tight control, robust induction and a highly titratable expression switch for inducible gene and microRNA expression studies. The system works through the CymR repressor that binds the cumate operator sequences (CuO) with high affinity. The repression is alleviated through the addition of Cumate, a non-toxic small molecule inducer that binds to CymR. This system has a dynamic inducibility, can be finely tuned and is reversible and inducible over and over for timed expression studies.
Lower Background than Other Systems
All-in-one Lentivector Formats
Fully Titratable 2000
AmpR
SV40 ORI SV40 poly-A
All-in-one Cumate Switch Inducible Lentivector CymR Puro
T2A
Cumate Switch Promoter
EF1a
MCS 5’NheI BstBI SwaI - 3’
pUC ori
SV40 ORI SV40 poly-A
SparQ™ All-in-one Cumate Switch Inducible IRES LentivectoR
TRA-60-1 T2A
WPRE
Puro
CymR EF1a
P GF
Cumate Switch Promoter
MCS 5’NheI BstBI SwaI NotI - 3’
Fluorescence Index (RFP+ Cells)
SparQ™
S
pUC ori
Cumate
IRE
AmpR
+ 50 mg/ml 1600
+ 10 mg/ml 800
+ 5 mg/ml
400
No Cumate 0
0
For more information: www.systembio.com/cumate -1-
+ 30 mg/ml
(CH3)2CHC6H4CO2H
1200
10
20
30
40
50
Cumate added (mg/ml)
CLONING AND VIRUS PACKAGING
The Cold Fusion Cloning Kit Next Generation Cloning Technology The Cold Fusion technology allows you to directly clone any PCR product(s) into any linearized expression vector, at any site without the use of restriction enzymes and ligase. Simply design primers with at least 15 bases of homology at the ends to direct the fusion location of the desired DNA fragment. Convenient one tube reaction, with a 5 minute incubation at room temperature followed by 10 minutes on ice. The Cold Fusion master mix prepares the DNA ends for sequence-directed alignment. The PCR product(s) rapidly and accurately fuse into the linearized vector in the desired location and orientation. Cold Fusion is so robust that multiple DNA fragments can be assembled simultanously and cloned into one construct in a single step. The system is highly efficient, with more than 95% positive cloning rates.
Broad PCR product size range with high cloning efficiency 500bp insert
1000bp insert 2000bp insert
COLD Fusion Cloning technology For more information, please visit: www.systembio.com/coldfusion
Easily Produce High Titer Lentivirus with SBI’s Tools System Biosciences (SBI) offers an extended set of cloning and expression lentivectors for efficient delivery of expression constructs in mammalian systems. To achieve highly efficient delivery and stable expression, the lentiviral constructs can be packaged in VSV-G pseudoviral particles and transduced into a wide range of cell lines or model organisms (mouse, rat, etc.). SBI has all the vectors and reagents you need to package, concentrate, titer and transduce lentiviruses into target cells efficiently. Once you have determined the titer of your virus, combine the appropriate amount of virus with TransDux and infect your target cells.
How to make high-titer virus pPACK™ Packaging Mix
Your Lentivector Construct
PureFection™
293TN Producer Cells
Cell Culture Medium Containing Viral Particles
5x PEG-it™ Solution
Global UltraRapid™ Lentiviral Titer Kit Measure Titer by qPCR
Packaging Plasmid(s)
16 14 y = 1.192x R2 = 0.999
12
Pseudoviral Particles
MOI
10
WPRE standards
8 6 4 2
pVSV-G
Negative control
Transfect 293TN Cells
Concentrate Virus
0 0
2
4
6
8
10
12
14
2-DDCt
For more information, please visit: www.systembio.com/lenti -2-
TRANSCRIPTION REPORTERS
Accurately Monitor Transcription Networks SBI’s lentiviral-based reporter system is a novel approach to study transcriptional regulation and offers many advantages over current transcription reporter systems. The activation of a signal transduction pathway (e.g. by growth factors, drugs, etc.) can be monitored by the expression level of the reporter gene controlled by a promoter containing the corresponding signal response elements. The number of reporter integrations can be controlled by varying the multiplicity of infection (MOI). Commonly used plasmid-based transcriptional reporter vectors often skew transcriptional network reporting due to their episomal nature. SBI’s lentivector-based transcription reporters integrate into the host’s genome and enable proper chromatinization to produce more faithful transcriptional activity reporting.
pGreenFire™ pGreenFire1 (pGF1) is a versatile HIV-based transcription reporter that co-expresses destabilized GFP and Firefly Luciferase enabling the simultaneous detection of GFP and Luciferase signals for quantitation of transcription activation. pGreenFire can be used with transcription response element repeats to monitor the activity of a particular transcription factor. Dual reporter system RSV 5’LTR
Ψ AmpR
p53-pGF Reporter (HT1080 cell line)
Multiple Cloning Site for TRE
0 mM Nutilin
10 mM Nutilin
Minimal CMV promoter is inactive without activation
pGreenFire Lenti-Reporter
pUC ori
TRE
TRE
TRE
TRE
mCMV
Transcription Factor Response Elements
Binding of Specific Transcription Factors to TRE sequences
BamHI
copGFP
SV40 ORI SV40 Poly-A
T2A peptide
3’ΔLTR WPRE
Luciferase Reporter
GFP Reporter
TRE
TRE
TRE
p53 pGF Reporter mCMV
GFP Reporter
Luciferase Reporter
Luciferase
Marke r EF1a
Optional: constitutive Puro or Neo Marker for Easy Cell Line Construction
Sample Luciferase data
TRE-specifc TRE-specifc TRE-specifc TRE-specifc Activator Activator Activator Activator
TRE
GFP
GFP
Transcription Factor Response Elements
Promoter is active when the specific Transcription factor is bound to the TRE sequences
RLU (x 1000)
cPPT Cla I EcoRI Xba I Spe I mCMV
3000 2000 1000
Control
1 uM
5 uM
10 uM
Nutilin
Available as ready-to-transduce virus or plasmid DNA Pathway
Transcription Factor
Wnt Wnt Hedgehog Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol
TCF/LEF1 cJun GLI ApoA1 Cyp7A1 ABCA1 ABCG1 ApoE CETP
+ TNFa Control GFP
Transcription Factor HIF-1 p53 AP1 CREB SMAD2/3/4 EGR1 NFkB STAT1 GAS
Phase
Pathway Hypoxia Cancer Cancer Cancer Cancer Cancer Inflammation Inflammation Inflammation
NFkB-pGF Reporter (293 cell line)
Many more reporters available, for more information visit: www.systembio.com/greenfire -3-
+1 ng/ml
+10 ng/ml
+100 ng/ml
MOLECULAR IMAGING VECTORS
Vivid Tracking of Subcellular and Cellular Activities Cyto-Tracers™ for Subcellular Tracking Molecular trafficking is a dynamic process in eukaryotic cells and the Cyto-Tracers provide the ability to light up cell compartments to monitor movement and localization of organelles and to trace endocytosis and exocytosis. SBI has created a line of lentivector-based Cyto-Tracers that utilize GFP-fusion proteins to mark cellular compartments, organelles, vesicles and structures to enable more long-term and more in-depth experimentation. Apoptosis induction can be monitored with the Caspase 3/7 activation Cyto-Tracer. All of the Cyto-Tracers can be used in transfections as well as packaged into virus to create stable GFP tracer cell lines in primary cells, tumor cell lines and stem cells.
RSV
Fusion Tag Directs Tracer Localization
5’LTR
Cat# CYTO118-PA-1
AmpR
Cat# CYTO113-PA-1
Peroxisomes
Cat# CYTO105-PA-1
Secretory Vesicles
Cytosol (Green)
GFP
pCT-CMV/MSCV GFP-Fusion-EF1-Puro
cPPT
Cat# CYTO119-PA-1
Tag
CMV or MSCV
pUC ORI
Nucleus
Cat# CYTO108-PA-1
Cat# CYTO111-PA-1
Microtubules Cytosol (Red) Plasma Membrane
SV40 ORI SV40 poly-A 3’Delta LTR WPRE
EF1 Puro
Cat# CYTO100-PA-1
Cat# CYTO112-PA-1
www.systembio.com/cytotracers Bioluminescent ExoQuick™ Imaging vectors (BLIV) for Cell Tracking
Molecular exosomeimaging techniques to visualize cell kinetics in small animals have resulted in an explosion in the precipitation knowledge of tumors, infectious diseases, and stem cell biology. The sensitivity and accuracy of in vitro and in vivo cell monitoring offers several advantages over traditional methods which involve animal sacrificing and histological analysis. Molecular imaging, for example, is normally non-invasive and allows for quantitatively assessing tumor growth and the effects of therapy over time. Molecular imaging technologies include bioluminescence imaging (BLI) and fluorescence imaging (FLI). BLI uses light generated from a luciferase enzyme-substrate and FLI uses red/green fluorescent protein (RFP or GFP) as a signal. SBI has created BLI and FLI dual reporter lentivectors and minicircle DNA constructs to perform molecular imaging in vivo.
Lentivector and Minicircle Reporter Options
Promoter choices • EF1 alpha • CMV • UbC • MSCV
Minicircle CMV-RFP-LUC
Lentivirus MSCV-GFP-LUC
Minicircle CMV-GFP-LUC
4 ug
2 ug
D-Luciferin reagent
250000
300000
200000
250000 200000
150000
RLU
• GFP-T2A-Luciferase • RFP-T2A-Luciferase
RLU
Reporter choices
100000
150000 100000
50000
50000
0 Control
Luciferase
0 Control
Luciferase
48 hr
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MINICIRCLE DNA TECHNOLOGY
Sustained Expression without Integration Minicircles are episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plasmid DNA backbone. Their smaller molecular size enables more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days. The Minicircle DNA elements are generated by an intramolecular (cis-) recombination from a parental plasmid (PP) mediated by PhiC31 integrase. The full-size MC-DNA construct is grown in a special host E. coli bacterial strain ZYCY10P3S2T. This highly engineered strain harbors an Arabinose-inducible system to express the PhiC31 integrase and the I-SceI endonuclease simultaneously. The ZYCY10P3S2T strain also contains a robust arabinose transporter LacY A177C gene. Adding arabinose to the media turns on the integrase and endonuclease genes. The PhiC31 integrase produces the MC-DNA molecules as well as PP-DNA from the full-size MC-DNA construct. The Sce-I endonuclease then degrades the PP-DNA backbone. Producing high quality minicircle DNA is made reliable with SBI’s MC-Easy™ Minicircle DNA production kit. The MC-Easy system enables the simple, reproducible and efficient way to produce high quality Minicircle DNA for your experiments. The finely tuned growth and induction media produce minicircle DNA that is free of parental and genomic DNA contamination. Minicircle expression lasts for weeks in vitro and in vivo.
MC-Easy Minicircle Production Kit
Expression cassette options SV40 poly-A
Promoter
Tr an sg en e
(switches on PhiC31 Integrase and I-Sce-I Endonuclease genes)
pU CO RI
+ Arabinose
4
M 1
Sample
2
M
attP
3 4
o
1 2 3
Parental Plasmid
SV40 poly-A
Avoid Remove Genomic Genomic & Parental Contamination Contamination
Lane
32x Sce-I Sites
ssion casse pre tte
Parental Plasmid attP
attB
Tra nsg ene
KanR
Promoter
Cat #
SV40 poly-A
Ex
KanR
32x I-Sce-I Sites (interspersed)
Minicircle
s ion pt
attB pUC ORI
Variety of Promoter and Marker Formats
attR
CMV MCS
MN501A-1
EF1a MCS
MN502A-1
CMV MCS EF1a
GFP
MN511A-1
CMV MCS EF1a
RFP
MN512A-1
EF1a MCS IRES
GFP
MN530A-1
EF1a MCS IRES
RFP
MN531A-1
CMV
GFP
positive control
CMV
RFP
positive control
MN602A-1
shRNA
CMV
GFP
H1
Genomic DNA
Genomic DNA Contamination MC-Easy Prep A Genomic & Parental DNA Contamination MC-Easy Prep B
MN601A-1
MNSI500A-1 shRNA
CMV
GFP
Puro H1
MNSI505A-1 shRNA
Parental DNA
EF1a
GFP
Puro H1
MNSI506A-1
Minicircle DNA
For more information, please visit: www.systembio.com/minicircle Minicircle transfections last for weeks Minicircles are easier to transfect into most cell types and expression can persist for weeks.
6 Hours
-5-
2 Days
4 Days
5 Days
6 Days
8 Days
PIGGYBAC TRANSPOSONS
Instant and Reversible Transgenesis The PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. SBI is a fully licensed provider of PiggyBac technologies from Transposagen, Inc.
PiggyBac Benefits
Cut and Paste Integrations
• One transfection makes transgenic cell lines
- PB Transposase
• Stable, heritable expression that is reversible
+ PB Transposase
GFP
GFP
Phase
Phase
• No cargo limit — integrate 10-100kb • Multiple integrations simultaneously • Effective in Human, Mouse and Rat cells Cat# CYTO100-PA-1
5’ PB Terminal Repeat Core Insulator
MCS
3’ PB Terminal Repeat Core Insulator SV40 poly-A
Promoter CMV CMV CMV CMV CMV MSCV
EF1
Marker
Catalog # PB510B-1 PB511B-1 PB512B-1 PB513B-1 PB514B-1 PB713B-1
PiggyBac Single Promoter Vector Formats
CMV or MSCV
Marker Puro GFP RFP GFP+Puro RFP+Puro GFP+Puro
3’ PB Terminal Repeat
5’XbaI NheI EcoRI BstBI SwaI BamHI NotI HindIII - 3’
Core Insulator SV40 poly-A
Marker
Catalog # Marker PB530A-1 PB531A-1 PB533A-1
GFP RFP Neo
5’ PB Terminal Repeat
AmpR
Core Insulator
pUC ori
3’ PB Terminal Repeat
ES
PiggyBac Dual Promoter Vector Formats
Core Insulator
pUC ori
PiggyBac shRNA Vectors
Puromycin selection (10g/ml) 3 days EF1
MCS 5’XbaI NheI EcoRI BstBI SwaI BamHI NotI HindIII - 3’
- PB Transposase
+ PB Transposase
RFP
RFP
Phase
Phase
EF1or CMV
Cat# PBSI505A-1 (CMV) Cat# PBSI506A-1 (EF1)
GFP
pUC ori
5’ PB Terminal Repeat
AmpR
IR
AmpR
Core Insulator EcoRI
ro T2A Pu
H1 BamHI
SV40 poly-A
For more information, please visit: www.systembio.com/piggybac -6-
STEM CELL RESEARCH
Xeno-free Culture Media & Potent Growth Factors PSGro® hESC/iPSC & MesenGro® Mesenchymal Stem Cell Growth Media PSGro is a fully defined xeno-free, human embryonic stem (ES) and induced plutipotent stem (iPS) cell culture medium that does not require feeder layers. PSGro enables the proper maintenance and expansion of pluripotent stem cells that sustain the correct morphology and expression of pluripotency markers. The medium supports robust proliferation with retention of a normal karyotype and differentation potential into multiple lineages across ectoderm, mesoderm and endoderm germ layers. MesenGro is a chemically-defined, serum-free and xeno-free medium to grow human mesenchymal stem cells (hMSCs). Maintain Stem Cell Morphology hESCs
Retain Stem Cell Marker Expression Oct4
hiPSCs
TRA-1-81
PSGro®
hESC/iPSC Growth Medium Accelerating discoveries through innovations
Basal Medium Serum-Free, Xeno-Free Feeder-Independent
catalog number: SC500M-BM
Store at 2 to 8 ºC For research use only empowering stem cell r&d
www.systembio.com 888.266.5066
Highly Potent Growth Factors SBI and StemRD have partnered to provide researchers access to new highly purified, bioactive growth and differentiation factors and stem cell growth media. All Growth Factors and media supplements are purified using novel and proprietary expression systems (Human and E. coli). This results in highly pure protein factors that are more active than other commercial products. Q/C bioassays are used to assess stem cell signaling pathways for activity.
Pre-made Viral and Nonviral iPS Cell Lines ALS iPS cell line
Nanog
SSEA3
Phase
AP stain
For more information: www.systembio.com/stemcell -7-
SBI offers Human and Mouse Induced Pluripotent Stem (iPS) Cell Lines with matched source fibroblasts, which were reprogrammed using standard retrovirus techniques and validated for pluripotency markers. SBI also offers two non-viral iPS Cell Lines: Protein-derived (piPSCs) – available exclusively from SBI – and Minicircle-derived (mciPSCs), both of which are fully characterized, karyotyped and differentiation-enabled iPSCs - without virus integration or genetic modifications. These pre-made iPS cells can be used to study differentiation, allowing research into the pathways and factors involved in fate specification.
Human Disease Model iPS Cell Lines Available • • • • • •
Type I Diabetes Autoimmune Model Type II Diabetes Metabolic Model Amyotrophic Lateral Sclerosis (ALS) Neurodegeneration Model Metachromatic Leukodystrophy (MLD) Polyneuropathy Model Muscular Dystrophy Muscle Development Model Glioblastoma Tumorigenic Model
STEM CELL RESEARCH
Efficiently Reprogram and Transdifferentiate Cells Reprogram adult cells into a pluripotent state using SBI’s iPSC factor expression systems. SBI offers retroviral, lentiviral, PiggyBac transposon and non-integrating Minicircle factors for reprogramming. SBI’s pre-mixed pool of retroviral particles provides the easiest method for reprogramming, and each ready-to-use lot is validated for high reprogramming efficiency. The 4-in-1 PiggyBac and Minicircle DNA systems offer cutting edge techniques for nonviral reprogramming of source cells. SBI’s reprogramming technologies enable you to efficiently derive novel patient-specific iPSCs, including cells which are free of chemical and transgenic elements for greater clinical relevance.
iPSC Technology Options • • • • •
SSEA4
Classic pooled OSKM packaged Retroviruses Nonviral, non-integrating Minicircle DNA LNSO and OSKM vectors Nonviral PiggyBac OSKM transposons for footprint-free iPSCs Minicircle, PiggyBac and Lentiviral miR-302bcad/367 constructs Nonviral, non-integrating iPSC factor mRNA transcripts
Generate iPS Cells
Minicircles Lin28 V
CM
Minicircle Reprogramer 2A
2A
NA
NO
G
SO X2
5’ ITR
MMLV Retrovirus Lentivirus
AmpR
pUC ori
PiggyBac Minicircle DNA RNA Small Molecules Protein
Adenovirus
3’ ITR Core Insulator
Core Insulator CMV promoter
Oct4
Human 4-in-1 2A Sox2 iPSC 2A Transposon 2A Klf4 Vector Myc 2A
SV40 poly-A
Puro
GFP
EF1 Promoter
Safety
Oc t4 So x2 Kl f4 cM yc Lin 28 Na no g
mRNAExpress™ Transcripts
Efficiency
4 OCT
GFP
MC-LGNSO
M ar ke GF r P
PiggyBac
Efficiency versus Safety iPSC Methods
2A
2A
NANOG
Direct Reprogramming Transdifferentiation
1987
2004 2006 2007 2008 2010 2010
Fibroblasts B cells
Fibroblasts
2010
2010
Fibroblasts Fibroblasts Fibroblasts B cells Fibroblasts Exocrine cells
Transdifferentiation (TD) is the direct conversion of one cell type to another. This reprogramming method provides a fast route for creating novel cell types and manufacturing Oct4 Pdx1 Ascl1 Pax5 Sox2 Oct4 Ngn3 Brn2 Oct4 CEBP functional tissues. The mRNAExpress™ transcript system can ablation MyoD Klf4 Gata4 MafA Myt1l Sox2 Myc Mef2c Klf4 produce mRNAs synthesized in vitro. The mRNAs have Tbx5 enhanced stability by incorporating modified nucleobases and offer increased translatability with 5' cap analogs and Muscle Macrophage iPSC Blood cells Beta cell Neuron Cardiomyocyte Blood cells Cardiomyocyte 3' polyA tails. The mRNAs can be delivered through transfection into a broad range of cell types, including fibroblasts and lymphocytes. This system enables a titratable, reproducible and non-integrating method for directly expressing key cellular factors involved in generating iPS cells as well as direct differentiation factors. SBI’s TD-Consortium includes collections of transcription factors and microRNA precursors built in constitutive or inducible lentivector and minicircle formats to allow you to harness TD technology and advance regenerative medicine research.
For more information: www.systembio.com/stemcell
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STEM CELL RESEARCH
Characterize and Monitor Pluripotency The pluripotent status of stem cells can be characterized by a high level of Alkaline Phosphatase (AP) activity, along with the expression of multiple pluripotency markers including the transcription factors Nanog, Oct4, Sox2, stage-specific embryonic antigens, SSEA-1, -3, -4, and tumor related antigens, TRA-1-60, TRA-1-81. SBI provides affinity purified and validated antibodies to detect the human pluripotent stem cell markers Oct4, Nanog, SSEA-3 and TRA-1-60. The antibodies are available individually, or can be purchased as a complete iPS verification kit that comes with all four antibodies plus an alkaline phosphatase staining kit. Alkaline phosphatase (AP) is a universal pluripotent marker for all types of pluripotent stem cells including embryonic stem cells, embryonic germ cells, and induced pluripotent stem cells. SBI offers AP staining kits, pluripotency antibody kits and pluripotency monitoring Promoter and Response element reporters to facilitate tracking stem cells.
Promoter and Response Reporters Oct4 • Nanog Promoter Reporters
Oct4 • Sox2 Response Reporters
hNanog
Oct4 CR4
Blue-Color AP Kit
Sox2 SRR2
Phase Contrast
hOct4
Pluripotency Marker Antibody and AP Kits
GFP Fluorescence
Oct4
Nanog
SSEA-3 H9 Human Embryonic Stem Cells
TRA-1-60
Red-Color AP Kit
Human iPS Cells
Track Differentiation with Lineage Reporters Cell-specific promoters drive GFP/RFP and Zeocin/TK markers in differentiating cells to allow monitoring of specification in real time. Trace differentiation across Neural, Hematopoietic, Myogenic, Structural and Endocrine lineages. These lentiviral reporters can be used to develop new directed differentiation protocols and to study cell fate specification. The dual function lenti-reporters are conveniently available as lentivector plasmids or ready-to-transduce lentiviral particles.
Differentiation Reporter Data
RSV HIV 5' LTR Ampr
pUC ori
Dual Function Lenti-reporters
SV40 ORI SV40 Poly-A 3’ΔLTR WPRE
Zeocin - or TK
-9-
T2A
Mouse Glial Fbrillary Acidic Protein Reporter
Mouse Troponin Reporter
Differentiation to Astrocytes from Stem Cells
Differentiation with Retinoic Acid
cPPT
–RA
+RA
Cell-specific Promoter GFP - or RFP
Astrocyte-specific GFAP promoter reporter data is Astrocytes shown above. Clearly identify specific cells within a mixed population. Only the astrocytes are GFP positive in a neural network including mature neurons and oligodendrocytes.
h9c2 rat cardiac myoblasts stably transduced with pGZ-mTnnt2 differentiation reporter and incubated in the presence or absence of retinoic acid for 2 days.
www.systembio.com/stemcell
RNAi LIBRARIES
GeneNet™ shRNA Pooled Lentiviral Libraries SBI’s pooled lentiviral libraries allow you to perform high-throughput screening studies on a genome-wide or pathway-focused basis. Pooled lentiviral libraries enable simultaneous identification of multiple genes or microRNAs that alter a specific cellular phenotype in a single experiment. Lentiviral libraries are available as prepackaged virus, so you can begin transducing cells the day you receive the library. System Biosciences GeneNet™ shRNA Libraries allow you to perform high-throughput gene knockdown Overview of Library studies on a genome-wide or pathway-focused Screening Protocol basis. GeneNet Libraries are proven, high performance screening tools for gene knockdown Transduction studies. Three to four shRNA sequences target each with lentivirus library mRNA transcript and are synthesized and cloned Infect into SBI’s shRNA lentivectors that provide superior Target Cells infection efficiciency across numerous cell types Treatment and models. Highly-specific and unique shRNA to induce phenotype sequences are designed to be compatible with Affymetrix® GeneChips. This design feature Treat/Select Cells provides the ability to hybridize the shRNA Selection effectors recovered after screens to GeneChips for for phenotype easy identification of the exact shRNA producing Recover Cells with the experimental phenotype. Cost-effective RNAi Specific Phenotype screens can be performed by any research group. Amplification
Available GeneNet™ Libraries
of effector sequences from selected cells
GeneNet: Genome-Wide shRNA Libraries HIV-based Libraries
Transcripts Targeted
No. of shRNAs
Human 50K
47,400
200,000
Mouse 40K
39,000
150,000
Hybridize to Affymetrix GeneChip®
FIV-based Libraries
Transcripts Targeted
No. of shRNAs
Human 50K
47,400
200,000
Mouse 40K
39,000
150,000
Analyze with statistical software
Sorted Cells 10000
GeneNet: Pathway Focused shRNA Libraries Targeted Genes
No. of shRNAs
Human Apoptosis
597
6,876
Human Kinase
897
10,453
Human Phosphatase
244
2,719
HIV-based Libraries
3
1
= New Target Identified
2 1000
= Enriched shRNAs = Unchanged shRNAs = Selected out shRNAs
100
Unsorted Cells
mRNA Targets Discovered
For more information: www.systembio.com/rnai-libraries
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MICRORNA AND LNCRNA PROFILING
Profile MicroRNAs & LncRNAs by qPCR Measure microRNAs by qPCR with QuantiMir™ & miRNome Profilers MicroRNA and siRNA expression analysis is made easy with the QuantiMir™ small RNA quantitation system. Generate qPCR-ready cDNA from total RNA for accurate and sensitive expression measurements. QuantiMir rapidly tags and converts all small RNAs into detectable cDNAs for qPCR —one cDNA synthesis required to quantitate any microRNA. Complete Human, Mouse and Rat qPCR Arrays - 100% miRBase compatible. Characterize microRNA signatures in stem cells, cancer cell lines and FFPE samples. Custom qPCR arrays are also available.
Capture More Expression Data with Genome-wide miRNome qPCR Profiler Arrays Human complete set
Rat complete set
Mouse complete set
www.systembio.com/mirnome
Quantitate Long Non-coding RNAs by qPCR Long non-coding RNAs (LncRNAs) and large intergenic non-coding RNAs (lincRNAs) are emerging as master regulators of embryonic pluripotency, differentiation, patterning of the body axis and promoting developmental transitions. LncRNAs are larger than 200 nucleotides in length and are pervasively expressed across the genome. LncRNAs maintain the commitment to specific cellular fates through modification and remodeling of chromatin at the epigenetic level. Dysregulated expression of lncRNAs has been shown to be associated with a broad range of diseases such as Alzheimer's, psoriasis and many cancers. Studying the expression patterns of lncRNAs will be a crucial method to understanding the roles they play in many model systems. SBI has built a sensitive, accurate and robust qPCR array that is Human Stem Cell and Cancer LncProfiler qPCR Array strand-specific to enable researchers to closely profile the expression changes in the top lncRNAs known to date. All of the lncRNAs on the qPCR array have validated primer sets for well-annotated lncRNAs that are registered in the lncRNA database created by Dr. John Mattick (www.lncrnadb.org). A
1
2
3
21A
7SK
7SL
4
- 11 -
Malat1 lncRNA Tm = 78˚C
BC200 lncRNA Tm = 80˚C
HOTAIR lncRNA Tm = 83.6˚C
IGF2AS
IPW
Jpx
Kcnq1ot1
E
Malat1
mascRNA
MEG3
MEG9
F
PR PCGEM1 antisense i SNHG5 SNHG6
G H
60
8
9
ANRIL
E2F4 antisense
EGO B
HAR1B
HOTAIR HOTAIRM1
KRASP1
EgoA
L1PA16
MER11C ncR-uPAR
10
11
anti-NOS2A antiPeg11 BACE1AS Evf1 and EVF2
Emx2os
GAS5
HOTTIP Hoxa11as HOXA3as HOXA6as
12
BC200
Gomafu HULC
p21
RoR
SFMBT2
VLDLR
LOC 285194
LUST
NDM29
NEAT1
Nespas
NRON
NTT
p53 mRNA
SAF
SCA8
snaR
SNHG1
SNHG3
SNHG4
Tsix
TUG1
UCA1
UM9-5
U6
No assay control
PTENP1
RNCR3
Sox2ot
PSF inhibiting RNA SRA
ST7OT
Y RNA-1
Zeb2NAT
Zfas1
TEA Tmevpg1 ncRNAs Zfhx2as 18S rRNA
PRINS
Xist
1
A
70
WT1-AS
7
DLG2AS
HAR1A
D
6
AK023948 Alpha 280 Alpha 250
DISC2
TncRNA RNU43
GAPDH LAMIN A/C
Mouse LncProfiler qPCR Array
80
Normalized Expression
Sample qPCR Assay Specificty Tests
Profile LncRNAs in Skin Cancer
5
Air
CAR DHFR Dio3os B Intergenic upstream H19 H19 i H19 antisense upstream C
Normal Cancer
50
B C D E F
40 30 20 10 0
www.systembio.com/LncRNA
G
2
3
4
5
6
7
8
9
10
11
12
AK028326AK082072 ATIA antiPeg11 B2 SINE AK007836 AK141205 Oct4 RNA CDR1Dlx1as Emx2os Evf2 Foxn2-as GAS5 Gomafu antisense Dio3os linc1242 mHOTAIR HOTTIP Hoxa11as IGF2AS Jpx Kcnq1ot1 LINC-Enah LINC1331 linc1368 linc1609- linc1609- linc1610- linc1610- linc1610Linc 1623 Linc1633 lincENC1 lincRNAlong short long medium short Cox2 Neat1 v1/ Neat1 v2/ isoform Malat1 mascRNA MEG3 MEG9 MSUR1 Msx1as MEN Men beta LXRBSV PINC 1Kb Recomb. RepA Otx2os PINC Pldi Rian Rmst RNCR3 isoform hot spot transcript
SCA8 (KLHL1-AS) Six3os
SNHG1
Vax2os1 VL30 RNAs WT1-AS
Adapt33 Air
BACE1AS
BC1
BGn-As
BORG
Gtl2-as
H19
H19 antisense
SNHG3
Xist
H
SNHG4
Y RNAs
SNHG5
Zeb2NAT
SNHG6
Zfas1
Zfhx2as
Sox2ot Mistral
SRA
Tsix
TUG1
RNU43 18S rRNA (snoRNA) GAPDH
Linc1612 linc1547 linc1582 lincRNAp21
lincRNALINC -MD1 Sox2
Nespas
Nkx2.2AS NRON Six3osclone9
No assay Beta Actin U6 snRNA control
Human Disease-related qPCR Array 2
3
4
A
21A
AAA1
aHIF
AK023948
ANRIL
B
CCND1 ANCR
1
CMPD
DD3
DGCR5
DISC2
5
6
7
8
9
anti-NOS2A BACE1AS BC017743 BC043430 DLG2AS
EGO
GAS5
GOMAFU
HOXA1AS HOXA3AS HOXA3AS HOXA6AS HOXA11AS AA489505 BI823151 BE873349 AK092154
10
11
12
BC200
BCMS
BIC
H19
H19-AS
HAR1A
C
HAR1B
HOTAIR
HOTAIRM1
HOTTIP
HULC
IPW
IGF2AS
D
KRASP1
L1PA16
LIT
LOC285194
LUST
LincRNAVLDLR
LincRNASFMBT2
MALAT1
MEG3
MER11C
NEAT1
NCRMS
E
NDM29
PANDA
PAR5
PCAT-1
PCAT-14
PCAT-29
PCAT-32
PCAT-43
PCGEM1
PR-AT2
PRINS
F
PTENP1
RMRP
ROR
SAF
SCA8
Sox2OT
SRA
ST7OT1
ST7OT2
ST7OT3
ST7OT4
Telomerase RNA
PSF inhibiting RNA
G
TMEVPG1 TU_001762 9
TUG1
UCA1
WT1-AS
Y1
Y3
Y4
Y5
ZEB2NAT
7SK
Negative control
H
7SLscRNA 5.8SrRNA
U87 scaRNA
U6 smRNA
ACTB
B2M
PGK1
GAPDH
HPRT1
RPL1A
RPL13A
GDC
MICRORNA ANALYSIS
Overexpress Human and Mouse MicroRNAs There are expected to be over 2,000 different microRNAs encoded in the human and mouse genomes and they function by either blocking translation of, or degrading, mRNA species corresponding to specific genes. While the number of verified human and mouse microRNAs are expanding, there is an increasing need for effective functional testing. SBI offers an extensive collection of microRNA precursors in lentiviral vectors that can be used to modulate the expression of their targeted mRNAs to study microRNA function. The lentiviral vector constructs can be packaged into pseudoviral particles and delivered to primary cells, stem cells, or other hard-to-transfect cell lines and can be used in vivo. SBI can package any construct into high titer lentivirus as a custom service for your studies. Each construct in SBI’s collection consists of the native stem loop structure and 200-400 base pairs of upstream and downstream flanking genomic sequence. This unique feature ensures that the microRNAs expressed from SBI’s constructs will be correctly processed in the cell into mature microRNA. www.systembio.com/lentimir RSV 5’LTR Ψ
Constitutive Human and Mouse Lenti-miRs
AmpR
Lenti-miRs pUC ORI
cPPT
Human (GFP) Mouse (GFP+Puro)
SV40 ORI SV40 poly-A 3’ΔLTR
CMV
EF1 GFP GFP+Puro
WPRE
Drosha processing
MicroRNA Precursors
The advantage of SBI’s constructs is that they can be used in transient transfection experiments as well as stably expressed in a wide variety of cell types, as opposed to synthetic microRNAs that can only be transiently expressed in cells. miRISC complexes
Dicer processing
5’ 3’
Dicer 5’
3’
AGO2 5’
3’
3’
5’
Targeted mRNA transcript
5’
Permanent MicroRNA Inhibition with miRZips™
l tro n Co
s es 9a r wn a p -2 o x d 9 e ck ip-2 er -miR o v O nti Kn iRZ e m L
Targeted protein Western blot probed with anti-Target protein antibodies
miRZip anti-sense microRNAs are stably expressed RNAi hairpins that have anti-microRNA activity. These miRZip shRNAs produce short, single-stranded anti-microRNAs that competitively bind their endogenous microRNA target and inhibit its function. The result is the derepression and elevation of the protein levels of the transcripts targeted by the microRNA being "zipped". Permanent MicroRNA Interference miRZip-145 Target mRNA de-repressed 5’ m7GpppG
microRNA-145 miRZip-145
miRZip-145 induces cMyc protein upregulation
Control
TRA-60-1
AAAA 3’
miRZip-145 TRA-60-1
c-Myc β-actin
Target protein
RISC
www.systembio.com/mirzips - 12 -
EXOSOME ISOLATION
One-step Exosome Isolation and Detection SBI developed two effective polymer-based methods to precipitate exosome microvesicles from serum, tumor ascites fluids, breast milk, cerebral spinal fluid, urine and tissue culture media samples. The polymer works by forming a mesh like network that captures microvesicles that range in size from 60 - 180 nm in diameter. SBI's ExoQuick is optimized for serum and ascites fluids and ExoQuick-TC™ exosome precipitation reagent is a distinct polymer formulation that has been engineered for exosome isolation from media and urine samples. This technology makes microRNA and protein biomarker discoveries simple, reliable and quantitative.
ExoQuick-TC ExoQuick -TC™
ExoQuick Benefits · · · · · ·
ExoQuick™ Optimized for serum
+
ExoQuick Solution
Exosome Antibody ExoAb Kits SBI offers individual antibodies for CD63, CD9, CD81 and Hsp70 as well as a Western blot sampler kit (Catalog# EXOAB-KIT-1) which includes four exosomal marker antibodies: CD63, CD9, CD81, HSP70 (rabbit anti-human) and also includes a goat anti-Rabbit IgG HRP conjugated secondary antibody specifically tested for use in exosomal protein analysis. 53 ) -7 0 kD
a)
Serum or Biofluid
No time-consuming ultracentrifugation Less expensive than costly antibody beads Compatible with biofluids from any species Isolated exosomes are intact and bio-active Recovers more exosomes than any other method Procedure takes less than an hour
(~
D6 aC 3 (~ D9 53 aC (~2 kDa D8 8 k ) aH 1 (~ Da) SP 26 70 kD a
ExoAbs
Exosome EM Analysis Incubate at 4˚C 30 minutes or overnight
aC
kDa MW 100 70 5525
Simple one-step precipitation
35 100nm
Exosome pellet
NANOSIGHT NANO
ma
90 nm
Exosome RNA Analysis
MW
Supernatant
NanoSight Particle Analysis 2.92 x 10^8 particles/ml
Spin in microfuge for 5 minutes
N a nopa rtic le T ra c king A na lys is ( N T A ) V e rs ion 2 . 1 BUILD 0329
rke Su r pe rn ata nt Ex oQ uic kP ell
et
Data coutesy of Dr. Vladimir Baranov (Umeå University, Sweden)
Nuc. 300 200
Small RNAs
100 0
100
200
300
400
500
Particle Size (nm)
600
700
800
900
15% PA RNA Gel
For more information, please visit: www.systembio.com/exoquick - 13 -
EXOSOME ANALYSIS
Measure Exosome Particles by ELISA Assays SBI’s ExoELISA™ kits are designed as a direct Enzyme-Linked ImmunoSorbent Assay (ELISA). The exosome particles and their proteins are directly immobilized onto the wells of the microtiter plate. After binding, wells are coated with a block agent to prevent non-specific binding of the detection antibody. The detection (primary) antibody is added to the wells for binding to a specific antigen (e.g. CD63) protein on the exosomes. ExoELISA kits come with exosome standards to make calibration curves.
Three primary antibody detection formats
Highly Sensitive and Quantitative ELISAs
OD450 nm
OD450 nm
0
5E+9 1E+10 # Exosome Particles
1.5E+10
CD81 Serum
0
5E+9 1E+10 # Exosome Particles
1.5E+10
ExoELISA Standards Calibrated by NanoSight
y=7E-11x + 0.124 R2 = 0.993
90
NANOSIGHT NANO N a nopa rtic le T ra c king A na lys is ( N T A ) V e rs ion 2 . 1 BUILD 0329
Concentration (particles 1x10^6)
For more information: www.systembio.com/exoelisa
y=6E-11x + 0.092 R2 = 0.992
OD450 nm
A Horseradish Peroxidase enzyme (HRP) linked secondary antibody (goat anti-rabbit) is used for signal amplification and to increase assay sensitivity. A colorimetric substrate (Extra-sensitive TMB) is used for the assay read-out. The accumulation of the colored product is proportional to the specific antigen present in each well. The results are quantitated by a microtiter plate reader at 450 nm absorbance and calibrated by the exosome standards provided in the kit. The exosome standards are provided in number of exosome particles as determined by NanoSight particle analysis measurements.
CD9 Serum
CD63 Serum y=5E-11x + 0.117 R2 = 0.971
0
5E+9 1E+10 # Exosome Particles
1.5E+10
0
100
200
300
400
500
600
700
800
900
Particle Size (nm)
Purify and Amplify Exosomal RNAs RNAs present in patient body fluids are a rich and untapped source of disease-related biomarkers. The RNAs are stable in serum because they are encapsulated in circulating exosomes. The SeraMir kit includes everything needed to accurately and sensitively measure RNAs from serum samples. Exosomes are efficiently isolated using SBI’s ExoQuick solution, and the exoRNAs are purified using a phenol-free lysis buffer and rapid spin columns. The SeraMir kit enables the 3’ tailing and simultaneous tagging of both 5’ and 3’ ends during cDNA synthesis—ready for qPCR. Primers for PCR amplification are included to make double-stranded cDNA that is ready to generate sense-strand exoRNAs using T7 IVT. Use the amplified exoRNAs for microarrays and NextGen sequencing applications. Isolate Exosomes
How the SeraMir Kit Works • Precipitate exosomes from patient biofluids with ExoQuick • Purify exoRNAs with SeraMir columns - bind, wash, elute • Tail and tag exoRNAs for qPCR with SBI’sTRA-60-1 microRNA qPCR arrays • Generate cDNA to make sense-strand T7 IVT RNA transcripts • Amplify exoRNAs for Microarrays and NextGen Sequencing
ExoQuick™ exosome precipitation
Purify exoRNAs
Amplify ExoRNA M Un Amp
500 bp 200 bp
Supernatant
SeraMir
100 bp
Amplified exoRNA
For more information: www.systembio.com/seramir - 14 -
265 North Whisman Rd. Mountain View, CA 94043 Telephone: 650-968-2200 Email: info@systembio.com www.systembio.com