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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

-4-

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 (10g/ml) 3 days EF1

MCS 5’XbaI NheI EcoRI BstBI SwaI BamHI NotI HindIII - 3’

- PB Transposase

+ PB Transposase

RFP

RFP

Phase

Phase

EF1or 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