PRODUCT BROCHUREPRODUCT BROCHURE

Spend less time making tools, and more time making discoveries

Vector Technologies • MicroRNA • Exosomes • Stem Cells • Libraries

Accelerating discoveries through innovations

Cover and p1.p14.ai 2 4/10/2012 4:22:09 PM

Inducible Cumate Switch VectorsThe 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.

LENTIVECTOR SYSTEMS

- 1 -

TRA-60-1

Cumate Switch Other Systems

+ C

um

ate

- C

um

ate

- Ind

uce

r + In

du

cer

Lower Background than Other Systems

All-in-one Lentivector Formats Fully Titratable

0 10 20 30 40 50

2000

1600

1200

800

400

0

Flu

ore

sce

nce

In

de

x (

RF

P+ C

ell

s)

Cumate added (mg/ml)

+ 5 mg/ml

+ 10 mg/ml

+ 30 mg/ml

+ 50 mg/ml

No Cumate

EF1a

pUCori

AmpR

All-in-one

Cumate Switch

Inducible

Lentivector MCS5’-

NheI

BstBI

SwaI

- 3’

CymR

PuroT2A

SparQ™ Cumate SwitchPromoter

SV40poly-A

SV40ORI

EF1a

WPRE

pUCori

AmpR

All-in-one

Cumate Switch

Inducible IRES

LentivectoR MCS5’-

NheI

BstBI

SwaI

NotI

- 3’

IRE

S

CymRPuro

GFPT2A

SV40poly-A

SV40ORI

Cumate SwitchPromoter

SparQ™

For more information: www.systembio.com/cumate

Eff icient 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(3) a producer cell line for packaging

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.

(CH3)2CHC6H4CO2H

Cumate

PROMOTER OPTIONS MARKER OPTIONS

CMV 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

NANO

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- 2 -

CLONING AND VIRUS PACKAGING

NANO

ExoQuick-TC

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 2000bp insert1000bp insert

COLD FusionFusionCloning technology

Easily Produce High Titer Lentivirus with SBI’s ToolsSystem 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.

For more information, please visit: www.systembio.com/lenti

For more information, please visit: www.systembio.com/coldfusion

How to make high-titer virus

293TNProducer Cells

Concentrate Virus

pPACK™Packaging Mix

YourLentivectorConstruct

Pseudoviral Particles

5x PEG-it™Solution

Cell Culture

Medium Containing

Viral Particles

PureFection™

WPREstandards

Negativecontrol 0 2 4 6 8 10 12 14

16

14

12

10

8

6

4

2

0

y = 1.192xR2 = 0.999

MO

I

2-DDCt

Global UltraRapid™ Lentiviral Titer Kit

Measure Titer by qPCR

pVSV-G

PackagingPlasmid(s)

Transfect 293TN Cells

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TRANSCRIPTION REPORTERS

- 3 -

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.

Accurately Monitor Transcription Networks

Dual reporter system

pGreenFireLenti-Reporter

BamHI

mCMVSpe I

Xba IEcoRI

Cla IcPPT

3’ΔLTRWPRE

SV40 ORI

pUC ori

AmpR

RSV 5’LTRΨ

SV40 Poly-AcopGFP

Luciferase

T2A peptide

Multiple

Cloning Site

for TRE

EF1aMarker

Optional: constitutive Puro or Neo

Marker for Easy Cell Line Construction

Binding of Specific Transcription Factors

Minimal CMV promoter is inactive without activation

mCMV

TRE-specifcActivator

mCMV GFP ReporterTRETRETRETRE

to TRE sequences

TRE-specifcActivator

TRE-specifcActivator

TRE-specifcActivator

TRETRETRETRE Luciferase Reporter

Luciferase Reporter

Transcription Factor Response Elements

Transcription Factor Response Elements

GFP Reporter

Promoter is active when the specific Transcription

factor is bound to the TRE sequences

p53-pGF Reporter (HT1080 cell line)

p53 pGF Reporter

0 mM Nutilin 10 mM Nutilin

1000

2000

3000

Control 1 uM 5 uM 10 uMNutilin

RL

U (

x 1

00

0)

GFP

Sample Luciferase data

GFP

Many more reporters available, for more information visit: www.systembio.com/greenfire

Phas

e

GFP

Control +1 ng/ml +10 ng/ml +100 ng/ml

+ TNFa

NFkB-pGF Reporter (293 cell line)

Pathway Transcription Factor Pathway Transcription FactorHypoxia HIF-1 Wnt TCF/LEF1 Cancer p53 Wnt cJun Cancer AP1 Hedgehog GLICancer CREB Cholesterol ApoA1Cancer SMAD2/3/4 Cholesterol Cyp7A1Cancer EGR1 Cholesterol ABCA1Inflammation NFkB Cholesterol ABCG1Inflammation STAT1 Cholesterol ApoEInflammation GAS Cholesterol CETP

Available as ready-to-transduce virus or plasmid DNA

p2.p13andp3.p12.ai 1 4/10/2012 4:26:21 PM

Vivid Tracking of Subcellular and Cellular Activities

MOLECULAR IMAGING VECTORS

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www.systembio.com/cytotracers

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.

ExoQuick™exosomeprecipitation

Lentivector and Minicircle Reporter Options

Bioluminescent Imaging vectors (BLIV) for Cell Tracking

Molecular imaging techniques to visualize cell kinetics in small animals have resulted in an explosion in the 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.

Promoter choices

• EF1 alpha

• CMV

• UbC

• MSCV

Reporter choices

• GFP-T2A-Luciferase

• RFP-T2A-Luciferase

D-Luciferin reagent

Nucleus

Microtubules

Plasma Membrane

Peroxisomes

Cytosol (Red)

Cytosol (Green) Secretory VesiclesCat# CYTO108-PA-1

Cat# CYTO112-PA-1

Cat# CYTO118-PA-1

Cat# CYTO119-PA-1

Cat# CYTO100-PA-1

Cat# CYTO111-PA-1

Cat# CYTO105-PA-1 Cat# CYTO113-PA-1

AmpR

3’Delta LTRWPRE

Puro

SV40 poly-A

cPPT

SV40 ORI

pUC ORI

RSV

CMV or

MSCV

EF1

5’LTR

pCT-CMV/MSCVGFP-Fusion-EF1-Puro

Tag

GFP

Fusion Tag DirectsTracer Localization

4 ug 2 ug

48 hr

Lentivirus MSCV-GFP-LUC Minicircle CMV-RFP-LUCMinicircle CMV-GFP-LUC

0

50000

100000

150000

200000

250000

300000

Control Luciferase

RL

U

0

50000

100000

150000

200000

250000

Control Luciferase

RL

U

p4.p11andp5.p10.ai 1 4/10/2012 4:31:23 PM

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.

MINICIRCLE DNA TECHNOLOGY

- 5 -

Sustained Expression without Integration

Minicircle transfections last for weeksMinicircles are easier to transfect into most cell types and expression can persist for weeks.

For more information, please visit: www.systembio.com/minicircle

MC-Easy Minicircle

Production Kit

Transg

ene

KanR

pUC ORI

SV40 poly-A

ParentalPlasmid

attP

Promoter

attB

attR

Transgene

Promoter

Minicircle

+ Arabinose

SV40 poly-A

32x I-Sce-ISites (interspersed)

(switches on PhiC31 Integrase

and I-Sce-I Endonuclease genes)

6 Hours 2 Days 4 Days 5 Days 6 Days 8 Days

pUC ORI

attP

attB

32x Sce-ISites Expression cassette options

ParentalPlasmid

SV40 poly-A

CMV MCS

MCSEF1a

CMV MCS GFP

CMV MCS RFP

EF1a

EF1a

MCS GFP

MCS RFP

IRES

IRES

CMV

EF1a

EF1a

CMV

GFP

RFP

positive control

positive control

MN501A-1

MN502A-1

MN511A-1

MN512A-1

MN530A-1

MN531A-1

MN601A-1

MN602A-1

Cat #

Expression cassette options

KanR

CMV GFP MNSI500A-1H1shRNA

CMV GFP MNSI505A-1H1shRNA

Puro

GFP MNSI506A-1H1shRNA

PuroEF1a

Variety of Promoter and Marker Formats

M 1 2 M 3 4

AvoidGenomic

Contamination

Minicircle DNA

Genomic DNA

Parental DNA

RemoveGenomic & Parental

Contamination

Lane Sample1 Genomic DNA Contamination

2 MC-Easy Prep A

3 Genomic & Parental DNA

Contamination

4 MC-Easy Prep B

p4.p11andp5.p10.ai 2 4/10/2012 4:32:21 PM

- 6 -

Cat# CYTO100-PA-1

Instant and Reversible TransgenesisThe 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.

For more information, please visit: www.systembio.com/piggybac

PiggyBac Benefits

• One transfection makes transgenic cell lines

• Stable, heritable expression that is reversible

• No cargo limit — integrate 10-100kb

• Multiple integrations simultaneously

• Effective in Human, Mouse and Rat cells

Cut and Paste Integrations

- PB Transposase + PB Transposase

GFP

Phase

GFP

Phase

Puromycin selection (10g/ml) 3 days

- PB Transposase + PB Transposase

RFP

Phase

RFP

Phase

MCS

SV40 poly-A

EF1

CoreInsulator

5’ PBTerminalRepeat

CoreInsulator

pUCori

3’ PBTerminalRepeat

AmpR

PiggyBac

Single Promoter

Vector Formats

Marker

5’-

XbaI

NheI

EcoRI

BstBI

SwaI

BamHI

NotI

HindIII

- 3’Catalog # MarkerPB530A-1 GFPPB531A-1 RFPPB533A-1 Neo

IRES

Promoter Catalog # MarkerCMV PB510B-1 PuroCMV PB511B-1 GFPCMV PB512B-1 RFPCMV PB513B-1 GFP+PuroCMV PB514B-1 RFP+PuroMSCV PB713B-1 GFP+Puro

MCS

CMV or MSCV

SV40 poly-A

EF1

CoreInsulator

5’ PBTerminalRepeat

CoreInsulator

pUCori

3’ PBTerminalRepeat

AmpR

PiggyBac

Dual Promoter

Vector Formats

Marker

5’-

XbaI

NheI

EcoRI

BstBI

SwaI

BamHI

NotI

HindIII

- 3’

SV40 poly-A

EF1or CMV

CoreInsulator

5’ PBTerminalRepeat

CoreInsulator

pUCori

3’ PBTerminalRepeat

AmpR

PiggyBac

shRNA Vectors

Cat# PBSI505A-1 (CMV)

Cat# PBSI506A-1 (EF1)

T2A

GFP

Puro

H1

BamHIEcoRI

PIGGYBAC TRANSPOSONS

p6.p9andp7.p8_ver3.ai 1 4/10/2012 5:10:57 PM

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).

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Xeno-free Culture Media & Potent Growth Factors

Maintain Stem Cell Morphology

hiPSCs

Retain Stem Cell Marker Expression

Oct4 TRA-1-81

Accelerating discoveries through innovations

PSGro® hESC/iPSC Growth Medium

Serum-Free, Xeno-Free

Feeder-Independent

Basal Medium

catalog number: SC500M-BM

Store at 2 to 8 ºCFor research use only

empowering stem cell r&d

www.systembio.com 888.266.5066

Highly Potent Growth FactorsSBI 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 LinesSBI 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.

hESCs

• 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

Human Disease Model iPS Cell Lines Available

For more information: www.systembio.com/stemcell

Nanog SSEA3

Phase AP stain

ALS iPS cell line

STEM CELL RESEARCH

p6.p9andp7.p8_ver3.ai 2 4/10/2012 5:10:10 PM

- 8 -For more information: www.systembio.com/stemcell

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.

Efficiently Reprogram and Transdifferentiate Cells

iPSC Technology Options• 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

Transdifferentiation

CMV

Lin28

GF

P

NANOGSOX2

MC-LGNSOMinicircle Reprogramer

OC

T4

2A

2A

2A 2A

Generate iPS Cells

SSEA4 NANOG

Minicircles

CMVpromoter

SV40 poly-A

CoreInsulator

5’ ITR

CoreInsulator

pUCori

3’ ITR

AmpR

Human 4-in-1iPSC

TransposonVector

Oct4

Sox2

Klf4

2A

Myc

EF1Promoter

2A

2A

GFP2A

Puro

PiggyBac

mRNAExpress™Transcripts

Sox2Klf4 cM

yc

Lin28

Nanog

Oct

4GFP

Mark

er

1987 2004 2006 2007 2008 2010 2010 2010 2010

Fibroblasts

Muscle Macrophage iPSC Blood cells Beta cell Neuron Cardiomyocyte Blood cells Cardiomyocyte

MyoDCEBP

Pax5

ablation

Oct4

Sox2

Klf4

Myc

Pdx1

Ngn3

MafA

Ascl1

Brn2

Myt1l Gata4

Mef2c

Tbx5

Oct4 Oct4

Sox2

Klf4

Fibroblasts FibroblastsFibroblasts

FibroblastsFibroblasts

B cells B cellsExocrine cells

Direct Reprogramming

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 functional tissues. The mRNAExpress™ transcript system can produce mRNAs synthesized in vitro. The mRNAs have enhanced stability by incorporating modified nucleobases and offer increased translatability with 5' cap analogs and 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.

Effi

cie

nc

y

Safety

Minicircle DNA

MMLV Retrovirus

Lentivirus

Adenovirus

RNA

PiggyBac

Small Molecules

Protein

Efficiency versus Safety iPSC Methods

STEM CELL RESEARCH

p6.p9andp7.p8_ver3.ai 2 4/10/2012 5:10:31 PM

Characterize and Monitor Pluripotency

- 9 -

www.systembio.com/stemcell

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.

Blue-Color AP Kit

Pluripotency Marker Antibody and AP Kits

TRA-1-60

Oct4

SSEA-3

Nanog

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.

GFP

- or -

RFP

Zeocin

- or -

TK

WPRE

RSV

cPPT

HIV 5' LTR

Dual Function

Lenti-reporters

T2A

Ampr

pUC ori

3’ΔLTRSV40 Poly-A

SV40 ORI

Cell-specificPromoter

Astrocyte-specific GFAP

promoter reporter data is

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.

Astrocytes

Differentiation Reporter Data

Mouse Troponin Reporter

+RA–RA

h9c2 rat cardiac myoblasts stably transduced with

pGZ-mTnnt2 differentiation reporter and incubated

in the presence or absence of retinoic acid for 2 days.

Differentiation with Retinoic Acid

Mouse Glial Fbrillary Acidic Protein Reporter

Differentiation to Astrocytes from Stem Cells

Red-Color AP Kit GF

P F

luo

resc

en

ceP

ha

se C

on

tra

st

hOct4 hNanog

Oct4 • Nanog Promoter Reporters

H9 Human Embryonic Stem Cells Human iPS Cells

Oct4 CR4 Sox2 SRR2

Oct4 • Sox2 Response Reporters

Promoter and Response Reporters

Track Differentiation with Lineage Reporters

STEM CELL RESEARCH

p6.p9andp7.p8_ver3.ai 1 4/10/2012 5:11:18 PM

Overview of Library

Screening Protocol

Treatmentto induce phenotype

Selectionfor phenotype

of effector sequencesfrom selected cells

InfectTarget Cells

Treat/Select Cells

Recover Cells with

Specific Phenotype

Transductionwith lentivirus library

10000

1000

100

Unsorted Cells

mRNA Targets Discovered

Analyze with statistical software

Hybridizeto Affymetrix GeneChip®

Sorted Cells

2

1

3

Amplification

= New Target

Identified

= Enriched shRNAs

= Unchanged shRNAs

= Selected out shRNAs

Available GeneNet™ Libraries

HIV-based Libraries

Transcripts Targeted

No. of shRNAs

Human 50K 47,400 200,000

Mouse 40K 39,000 150,000

HIV-based LibrariesTargeted

GenesNo. of

shRNAs

Human Apoptosis 597 6,876

Human Kinase 897 10,453

Human Phosphatase 244 2,719

FIV-based Libraries

Transcripts Targeted

No. of shRNAs

Human 50K 47,400 200,000

Mouse 40K 39,000 150,000

GeneNet: Genome-Wide shRNA Libraries

GeneNet: Pathway Focused shRNA Libraries

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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

For more information: www.systembio.com/rnai-libraries

studies on a genome-wide or pathway-focused basis. GeneNet Libraries are proven, high performance screening tools for gene knockdown studies. Three to four shRNA sequences target each mRNA transcript and are synthesized and cloned into SBI’s shRNA lentivectors that provide superior infection efficiciency across numerous cell types and models. Highly-specific and unique shRNA sequences are designed to be compatible with Affymetrix® GeneChips. This design feature provides the ability to hybridize the shRNA effectors recovered after screens to GeneChips for easy identification of the exact shRNA producing the experimental phenotype. Cost-effective RNAi screens can be performed by any research group.

p4.p11andp5.p10.ai 2 4/10/2012 4:31:54 PM

Capture More Expression Data with Genome-wide miRNome qPCR Profiler Arrays

Profile MicroRNAs & LncRNAs by qPCR

MICRORNA AND LNCRNA PROFILING

- 11 - www.systembio.com/LncRNA

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.

Profile LncRNAs in Skin Cancer

Cancer

No

rma

lize

d E

xp

ress

ion

0

10

20

30

40

50

60

70

80

Normal

1 2 3 4 5 6 7 8 9 10 11 12

A 21A 7SK 7SL Air AK023948 Alpha 280 Alpha 250 ANRIL anti-NOS2A antiPeg11 BACE1AS BC200

BCAR

Intergenic DHFR

upstream i

Dio3os DISC2 DLG2ASE2F4

antisenseEgoA EGO B Emx2os

Evf1 and EVF2

GAS5 Gomafu

C H19H19

antisenseH19

upstream HAR1A HAR1B HOTAIR HOTAIRM1 HOTTIP Hoxa11as HOXA3as HOXA6as HULC

D IGF2AS IPW Jpx Kcnq1ot1 KRASP1 L1PA16 p21 RoR SFMBT2 VLDLRLOC

285194 LUST

E Malat1 mascRNA MEG3 MEG9 MER11C ncR-uPAR NDM29 NEAT1 Nespas NRON NTT p53 mRNA

F PCGEM1PR

antisense i

PRINS PSF inhibiting

RNAPTENP1 RNCR3 SAF SCA8 snaR SNHG1 SNHG3 SNHG4

G SNHG5 SNHG6 Sox2ot SRA ST7OT TEA ncRNAs

Tmevpg1 TncRNA Tsix TUG1 UCA1 UM9-5

H WT1-AS Xist Y RNA-1 Zeb2NAT Zfas1 Zfhx2as 18S rRNA RNU43 GAPDH LAMIN A/C U6No assay control

Human Stem Cell and Cancer LncProfiler qPCR Array

1 2 3 4 5 6 7 8 9 10 11 12

A Adapt33 Air AK007836 AK141205 AK028326-Oct4 AK082072 ATIA antiPeg11 B2 SINE

RNA BACE1AS BC1 BGn-As

B BORG CDR1-antisense Dio3os Dlx1as Emx2os Evf2 Foxn2-as GAS5 Gomafu Gtl2-as H19 H19

antisense

C mHOTAIR HOTTIP Hoxa11as IGF2AS Jpx Kcnq1ot1 linc1242 LINC-Enah LINC1331 linc1368 Linc1612 linc1547 linc1582

D linc1609-long isoform

linc1609-short

linc1610-long

linc1610-medium

linc1610-short Linc 1623 Linc1633 lincENC1 lincRNA-

Cox2lincRNA-p21

lincRNA-Sox2 LINC -MD1

E LXRBSV Malat1 mascRNA MEG3 MEG9 MSUR1 Msx1as Neat1 v1/ MEN

Neat1 v2/ Men beta Nespas Nkx2.2AS NRON

F Otx2os PINC PINC 1Kb isoform Pldi Recomb.

hot spot RepA transcript Rian Rmst RNCR3 SCA8

(KLHL1-AS) Six3os Six3os-clone9

G SNHG1 SNHG3 SNHG4 SNHG5 SNHG6 Sox2ot SRA Tsix TUG1 Vax2os1 VL30 RNAs WT1-AS

H Xist Y RNAs Zeb2NAT Zfas1 Zfhx2as Mistral 18S rRNA RNU43 (snoRNA) GAPDH Beta Actin U6 snRNA No assay

control

Mouse LncProfiler qPCR Array

1 2 3 4 5 6 7 8 9 10 11 12

A 21A AAA1 aHIF AK023948 ANRIL anti-NOS2A BACE1AS BC017743 BC043430 BC200 BCMS BIC

BCCND1 ANCR CMPD DD3 DGCR5 DISC2 DLG2AS EGO GAS5 GOMAFU H19 H19-AS HAR1A

C HAR1B HOTAIR HOTAIRM1 HOTTIP HOXA1AS AA489505

HOXA3AS BI823151

HOXA3AS BE873349

HOXA6AS AK092154 HOXA11AS HULC IPW IGF2AS

D KRASP1 L1PA16 LIT LOC285194 LUST LincRNA-VLDLR

LincRNA-SFMBT2

MALAT1 MEG3 MER11C NEAT1 NCRMS

E NDM29 PANDA PAR5 PCAT-1 PCAT-14 PCAT-29 PCAT-32 PCAT-43 PCGEM1 PR-AT2 PRINSPSF

inhibiting RNA

F PTENP1 RMRP ROR SAF SCA8 Sox2OT SRA ST7OT1 ST7OT2 ST7OT3 ST7OT4 Telomerase RNA

G TMEVPG1 TU_0017629

TUG1 UCA1 WT1-AS Y1 Y3 Y4 Y5 ZEB2NAT 7SK Negativecontrol

H 7SL scRNA 5.8S rRNA U87 scaRNA U6 smRNA ACTB B2M PGK1 GAPDH HPRT1 RPL1A RPL13A GDC

Human Disease-related qPCR Array

BC200

lncRNA

Tm = 80˚C

HOTAIR

lncRNA

Tm = 83.6˚C

Malat1

lncRNA

Tm = 78˚C

Sa

mp

le q

PC

R A

ssa

y S

pe

cifi

cty

Te

sts

Mouse complete setHuman complete set Rat complete set

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 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).

Quantitate Long Non-coding RNAs by qPCR

www.systembio.com/mirnome

p4.p11andp5.p10.ai 1 4/10/2012 4:30:58 PM

MICRORNA ANALYSIS

- 12 -

TRA-60-1

Permanent MicroRNA

Inhibition with miRZips™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

Constitutive Human and Mouse Lenti-miRs

Overexpress Human and Mouse MicroRNAsThere 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.

The advantage of SBI’s constructs is that they can be used in transient transfec-tion 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.

www.systembio.com/lentimir

Targetedprotein

ControlOverexpress

Lenti-miR-29a

Knockdown

miRZip-29a

Western blot probed with anti-Target protein antibodies

RISC

AAAA 3’5’ m7GpppG

Target mRNAde-repressed

microRNA-145miRZip-145

miRZip-145

Target protein

TRA-60-1

β-actin

Control miRZip-145

c-Myc

miRZip-145 induces cMyc protein upregulation

www.systembio.com/mirzips

Droshaprocessing 5’

3’

5’ 3’5’3’

DicerAGO2

AmpR

3’ΔLTRWPRE

GFPGFP+Puro

SV40 poly-A

cPPT

SV40 ORI

pUC ORI

RSV

CMV

EF1

5’LTRΨ

Dicerprocessing

MicroRNAPrecursors

Lenti-miRsHuman (GFP)Mouse (GFP+Puro)

miRISC complexes

5’

5’

3’

Targeted mRNAtranscript

p2.p13andp3.p12.ai 1 4/10/2012 4:24:52 PM

EXOSOME ISOLATION

- 13 -

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 Benefits

· 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

For more information, please visit: www.systembio.com/exoquick

ExoQuick™

Optimized for serum

+Serum or Biofluid

Supernatant

Exosome pellet

ExoQuickSolution

Simple one-step

precipitation

Incubate at 4˚C 30 minutes orovernight

Spin in microfugefor 5 minutes

Super

natan

t Ex

oQuick Pe

llet

MW

mar

ker

300200

100

Nuc.

SmallRNAs

15% PA RNA Gel

Exosome RNAAnalysis

kDa

55

70

35

25

100MW aCD

63 (~53 k

Da)

aCD9 (~

28 kD

a)

aCD81 (~

26 kD

a)

aHSP70 (~

53-70 k

Da)

ExoAbs Exosome EM Analysis

100nm

Data coutesy of Dr. Vladimir Baranov (Umeå University, Sweden)

Particle Size (nm)

0 100 200 300 400 500 600 700 800 900

90 nm

BUILD 0329 1.2 noisreV )ATN( sisylanA gnikcarT elcitraponaN

NANONANOSIGHT

2.9

2 x

10

^8

pa

rtic

les/

ml

NanoSight Particle Analysis

ExoQuickExoQuick-TC-TC™

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.

COLD Fusion

p2.p13andp3.p12_ver2.ai 2 4/10/2012 4:29:33 PM

EXOSOME ANALYSIS

- 14 -

Measure Exosome Particles by ELISA AssaysSBI’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.

TRA-60-1

Highly Sensitive and Quantitative ELISAs

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.

OD

450

nm

# Exosome Particles

OD

450

nm

# Exosome Particles

OD

450

nm

# Exosome ParticlesParticle Size (nm)

0 100 200 300 400 500 600 700 800 900

90

BUILD 0329 1.2 noisreV )ATN( sisylanA gnikcarT elcitraponaN

NANONANOSIGHT

Co

nce

ntr

ati

on

(p

art

icle

s 1

x1

0^

6)

ExoELISA StandardsCalibrated by NanoSight

CD63 Serum CD9 Serum

CD81 Serum

0 5E+9 1E+10 1.5E+10 0 5E+9 1E+10 1.5E+10

0 5E+9 1E+10 1.5E+10

y=7E-11x + 0.124

R2 = 0.993

y=5E-11x + 0.117

R2 = 0.971

y=6E-11x + 0.092

R2 = 0.992

For more information: www.systembio.com/exoelisa

Three primary antibody detection formats

Purify and Amplify Exosomal RNAsRNAs 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.

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’s microRNA qPCR arrays • Generate cDNA to make sense-strand T7 IVT RNA transcripts• Amplify exoRNAs for Microarrays and NextGen Sequencing

Supernatant

Purify exoRNAs

Isolate Exosomes Amplify ExoRNA M Un Amp

100 bp

200 bp

AmplifiedexoRNA

500 bp

ExoQuick™exosomeprecipitation

SeraMirFor more information: www.systembio.com/seramir

Cover and p1.p14.ai 1 4/10/2012 4:21:15 PM

PRODUCT BROCHURE

265 North Whisman Rd.

Mountain View, CA 94043

Telephone: 650-968-2200

Email: info@systembio.com

www.systembio.com

Cover and p1.p14.ai 2 4/10/2012 4:22:51 PM