Thermo Scientific Open BiosystemscDNAs and ORFs

World's largest collection of pre-made cDNAs and ORFs

Fully sequenced, easy-to-transfer and cost-effective

Genome-scale and expression-ready collections

Genome-scaleCoverage Viral Format

Pre-made cDNAs and ORFs as tools for gene expression

Thermo Scientific Open Biosystems pre-made cDNA and ORF collections

Gene expression solutions and formats

Thermo Scientific Open Biosystems cDNA collections

The importance of full insert sequencing

Thermo Scientific Open Biosystems Open Reading Frame (ORF) collections

Thermo Scientific Open Biosystems Precision LentiORFs

Pre-defined cDNA and ORF gene families

Delivery and viral packaging

Gene Search and Online BLAST

References

• Fully sequenced cDNAs

• Gateway-adapted ORFs

• Expression-ready ORFs

• Gateway®-adapted ORFs

• Expression-ready cDNAs

• Lentiviral ORFs

• Lentiviral ORFs

• Expression-ready cDNAs

Expression-ready Easy-to-transfer

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Table of contents

The world’s largest and most complete collections of pre-made, full-length, sequence-verified cDNA and open reading frame (ORF) clones for reliable gene over-expression

ORF clones are derived from cDNA clones by removing the untranslated region (UTR) leaving just the protein coding sequence known as the ‘Open Reading Frame’ or ORF. The UTRs are replaced by recombinational cloning sites (att sites) for easy transfer.

To be "full-length", a cDNA clone must contain the complete open reading frame (ORF) and typically includes part or all of the 5' and 3' untranslated regions (UTRs).

cDNA clones provide the user with the protein coding sequence with its native stop codon and regulatory elements often found in the UTRs. By replacing the UTRs and stop codon with Gateway sites, the ORF clones offer increased flexibility when transferring to expression vectors.

Pre-made cDNAs and ORFs are versatile tools for gene analysis and protein science

Pre-made cDNA and ORF collections facilitate applications across many fields of biological research

cDNA and ORF clones provide the strongest evidence for the structure of RNA transcripts and are indispensible tools for determining gene function.

Thermo Scientific Open Biosystems products include the world’s largest collection of tools for gene analysis. These include fully sequenced cDNA clones for native protein expression, ORF clones that can be transferred to your expression vector of choice, and expression-ready lentiviral ORFs providing the fastest path to your gene analysis results.

cDNAs and ORFs offer experimental solutions for both cell biologists and protein scientists. cDNAs mirror the native RNA sequence including the protein coding region and the regulatory sequences often found in the UTR.

The non-coding regions are removed in the ORF clones and replaced with flexible cloning sites. This facilitates a variety of experiments including: tracking subcellular localization, protein-protein interaction studies, purification for in vitro assays or antibody production.

Full-Length cDNA Collections

cDNA

ORF

Full-Length ORF Collections

5’ UT R ORF5’UTR ORF 3’UTR

ORF att att

Native Protein UTR Biology

Fusion TagsRobust Expression

PhenotypicAnalysis

Subcellular Localization

RNAi HitValidation

CellBiology

AntibodyProduction

ProteinInteraction

In vitro Assays

ProteinScience

5’UTR ORF 3’UTR

ORF att att

5’UTR ORF 3’UTR

ORF att att

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Fully sequenced, easy-to-transfer and expression-ready cDNAs and ORFs

Steps involved in de novo cloning versus ordering pre-made cDNA or ORF clones.

Full-Length cDNA Collections

Mammalian Gene Collection (MGC)

• High-quality human, mouse and rat clones

• Fully sequenced and verified to contain a complete coding sequence (CDS)

Incyte Clone Collection

• Supplements MGC gene coverage

• Provides additional unique gene coverage and splice variants

• Exclusive to Thermo Scientific

Expression-ready MGC subset

• Fully sequenced

• Robust CMV promoter

Gateway-adapted ORF Collections

ORFeome Collaboration Collection

• Fully sequenced

• With or without stop codon

Human ORFeome

• Genome-scale coverage

• Derived from fully sequenced clones from the MGC

Precision LentiORFs

• Expression-ready lentiviral ORFs

• High-titer purified lentiviral particles for hard-to-transfect cell lines

Determine your gene of interest

Harvestcells

RNApurification

cDNAsynthesis

Gene-specific PCR

Gel sizing and

purification

Ligate and transform

Screen and fully sequence

insert

Gene search

Full-length clonedelivered

Start your experiment in

DAYS

Start your experiment in

WEEKS

Save time and money with pre-made cDNAs and ORFs

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cDNA ORFs LentiORFs

MGC Incyte ORFeome Collaboration Human ORFeome Precision LentiORFs

Species

Fully sequenced Yes Yes Yes Yes*

Expression-ready Subsets Yes

Easy-to-transfer Yes Yes

Fluorescent marker Yes

Ready-to-use Yes

Genome-scale Yes Yes Yes Yes Yes

Format

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Bacterial glycerol stock: Bacterial culture transformed with a plasmid vector, grown in presence of glycerol for freezer storage and supplied in single tubes.

Arrayed format: Bacterial glycerol stocks, custom arrayed in 96-well microtiter plates.

Arrayed library arrayed format: Genome-scale libraries of bacterial glycerol stocks arrayed in 96-well microtiter plates.

Gene families and pathways: Bacterial glycerol stocks of cDNA and ORF pre-defined libraries arranged by gene family and/or function and arrayed in 96-well microtiter plates.

Fluorescent marker: TurboGFP as marker of expression.

High-titer viral particles: Transduction-ready, high-titer (108 TU/mL) lentiviral particle format. Turnaround time is 6-8 weeks.

Precision LentiORF Starter Kits: All reagents and protocols necessary to start a gene expression experiment are provided in a single, convenient package.

Guaranteed** to express.

Format Format

Species

Mouse Human Rat

GFP

GFP GFP

GFP

GFP

Which gene expression solution is suitable for your research?

Follow the icons to your perfect product solution.

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** When used as part of a LentiORF starter kit according to kit protocols.

* Fully sequenced ORFs from the ORFeome collaboration.

*Guaranteed to match Genbank published sequence

Genome-scale coverage for Human, Mouse and Rat At the onset of the ‘Genomics Revolution’ the National Institutes of Health (NIH) spearheaded the creation of genome-scale cDNA collections for the human, mouse and rat. In 2009, this multi-institute project was completed, creating the most extensive, rigorously sequenced collection of mammalian cDNAs (<1 base in 50,000 nucleotide error rate)1,2,3.

• Pre-made genome-scale collections

• Sequence-verified to contain a complete ORF

• Guaranteed sequence*

Over 27,000 additional fully sequenced clones add depth and breadth to the genome-scale MGC cDNA resource.

• Provides rare gene coverage not included in the MGC collection

• Use our BLAST search to find cDNAs not covered by other collections

Mammalian Gene Collection (MGC) -setting the standard for gene expression

Human Mouse Rat

MGC cDNA clones 30,039 27,457 6,769

MGC Non-redundant genes

17,421 17,285 6,363

Human

Incyte Collection clones

27,000

To be "full-length", a cDNA clone must contain the complete open reading frame (ORF) and typically includes part or all of the 5' and 3' untranslated regions (UTRs).

Number of clones and unique genes covered by the MGC (A), and the number of additional Incyte clones (B) providing supplemental transcript variants.

A)

B)

5’UTR ORF 3’UTR

ORF att att

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Availability

1. Individual clone- glycerol stock

2. Libraries: Pre-defined gene families or custom-glycerol stock

3. Genome-scale library- glycerol stock

Genome-scale cDNA collections for native gene expression

Exclusive supplemental gene coverage is offered through the Incyte cDNA Collection

GFP

45% Putative full-length clone failure rate in the MGC pipeline

Cumulative numbers for putative and confirmed full-length clones over the history of the MGC (from the MGC weekly status report, August 28, 2005).

Alternative splicing events results in two variant cDNAs for the DNMT3B gene. (a) The wild-type transcript produces the full-length protein. (b) The variant occurs when an aberrant splicing event excludes exon 10 and retains intronic sequence. Exons outside of this area are unaffected, and the two isoforms would be indistinguishable via end sequencing and gel electrophoresis.

The importance of full-insert sequencing

Following full-insert sequencing, approximately 45% of putative full-length clones in the MGC pipeline were eliminated for various reasons including incomplete ORFs, chimeric sequences, and frameshifts.

The wide-spread phenomenon of alternative splicing provides further motivation for full-insert sequencing of cDNA clones. It is estimated that 74% of multi-exon genes are alternatively spliced4. Thus, clones that are only end-sequenced have a significant risk of being assigned an incorrect reference accession.

Multiple transcript variants of the DNA methylase gene (DNMT3B) can be expressed from a single cell line5. Despite only small transcript size differences these variants have dramatic effects on cells. Expression of DNMT3B7 has been shown to change gene expression profiles and alter genome methylation states of HEK293 cells to mimic those seen in cancer cell types. Without full-insert sequencing this splice variant would be indistinguishable from the wild- type transcript.

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b.

40,000

Putative Confirmed

30,000

20,000

10,000

0

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The ORFeome Collaboration has created a collection of fully sequenced ORFs that will represent all curated genes in the human genome. These clones are verified to have a full-length ORF and are easily transferable into any Gateway-adapted expression vector. Additional clones for this expanding collection are offered as they become available.

• Fully sequenced

• Easy-to-transfer - Gateway-adapted

• Native and fusion-ready options – with or without stop codon

Visit www.orfeomecollaboration.org for more details.

The Human ORFeome Collection was developed by Dr. Marc Vidal’s lab at the Dana-Farber Cancer Institute. Using the MGC cDNA collection6,7,8 as templates, these ORFs were created by high- throughput PCR providing broad gene coverage. The resulting PCR product was cloned into an entry vector and was confirmed to contain the correct MGC insert via 5’ end sequencing read.

• Broad gene coverage

• Easy-to-transfer – Gateway-adapted

• Maximum flexibility – Stop codon removed to allow fusion-tags

ORF clones are derived from cDNA clones by removing the untranslated region (UTR) leaving just the protein coding sequence known as the ‘Open Reading Frame’ or ORF.

Gateway cloning provides fast and efficient gene content portability without the need for PCR or re-sequencing. This allows flexible cloning strategies to quickly incorporate the cloned ORF into many different experimental designs.

5’UTR ORF 3’UTR

ORF att att

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Entry Clone

Destination Clone

Expression Clone

PurificationOver-

Expression

Localization

ccDB

ORF

att att

att att

ORFatt att

Human ORFeome Collection

The ORFeome Collaboration Collection (OCC)

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Availability

1. Individual clone- glycerol stock

2. Libraries: Pre-defined gene families or custom-glycerol stock

3. Genome-scale library- glycerol stock

Genome-scale Open Reading Frame (ORF) collections for robust over-expression

GFP

The Precision LentiORF Collection is a library of full-length ORFs in an expression-ready lentiviral vector generated from the fully sequenced ORFeome Collaboration Collection. These ready-to-use ORFs are high-quality, versatile and guaranteed* to express.

• Expanded delivery options – use as plasmid or viral particles for hard-to-transfect cells

• Efficient expression - even at low multiplicity of infection (MOI)

• Flexible applications - cloning site for adding a custom fusion-tag

HEK293T cells transduced at a range of MOIs. Western blot analysis depicts the expected increase in protein expression correlating with an increase in the number of LentiORF viral particles applied to the cell.

Controlled ORF expression levels with increasing viral titers.

MOI MOI MOI MOI

1 3 10 20

Viral particles per cell

ORF

Tubulin

Precision LentiORF transduced HEK293T cells expressing nuclear localized TurboGFP (TurboGFPnuc).

Easily track transduction and expression.

Straightforward for easy to transfect cells

Transfection starter kit

2 ORF clones - Glycerol

RFP control - Glycerol

2 mL Express-In transfection reagent

Create your own lentiviral particles for hard to transduce cells

Transduction starter kit

2 ORF clones - Glycerol

RFP control - Glycerol

2 mL Express-In transfection reagent

Trans-Lentiviral packaging mix (10 rxn)

Transduction-ready high-titer lentiviral particles

Transduction starter kit

2 ORF clones - high-titer virus

4x25 µL @ 1x108 TU/mL

RFP control - high-titer virus

1x25 µL @ 1x108 TU/mL

www.thermo.com/GeneExpression 8

Availability

1. Individual clone- glycerol stock

2. Individual clone - lentiviral particles**

3. LentiORF starter kits

4. RFP LentiORF control***

5. Genome-scale library- glycerol stock

pLOC

-2a-ORF

Ampr r

IRES

CMV

Ψ cPPTBlastr

WRE

5’LT

R

sinLTR

pUC sv40 Ori

tGFP(nuc)

Multi TagCloning Site

Expression-ready lentiviral ORFs for guaranteed expression

Precision LentiORF Collection

Vector Element Utility

CMV Promoter Efficient expression

TurboGFP(nuc) Visual tracking of transduction andexpression

MultiTag Cloning Site Convenient cloning site for the additionof a purification or tracking tag

Blasticidinr Selection for pure populations

SIN LTR 3' Self-inactivating long terminal repeatprovides additional safety feature

GFP

* When used as part of a LentiORF starter kit according to kit protocols. ** 6-8 weeks for delivery *** Serves as positive or negative control

The Druggable Genome set is a focused sub-set of the MGC collection consisting of genes considered to be candidates for therapeutic development. The resulting proteins from these cDNAs are predicted to be targets for small molecules. As a cDNA collection, it can be transferred to an expression vector for over-expression screening or for protein production in screening validations9,10.

Druggable-Genome:

GPCR Ion Channel Kinases Peptidase Phosphatases Ubiquitin conjugation

Pre-defined cDNA and ORF gene family sub-libraries have been assembled using innovative bioinformatic tools to reflect the latest genetic and ontological information from peer-reviewed publications and data-bases. These collections represent the most recent publications, providing the highest confidence for your over-expression experiments.

Please visit www.thermo.com/genefamilies for more information or contact openbiosystems@thermofisher.com to build your custom gene set.

The Druggable Genome set

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Availability

1. Libraries: Pre-defined and custom, arrayed in 96-well plate format- glycerol stock

All Gene Families

Cell Cycle Oxygen Binding

Cytokine Peptidase

GPCR Phosphatases

Growth Factor Plasma Membrane

Insulin Receptor Signaling Protein Kinase

Ion Channel Transcription Factor

Kinases Transporter

Nuclear Receptor Ubiquitin conjugation

Pre-defined gene family cDNA and ORF librariesfor targeted functional studies

cDNA and ORF Libraries arranged by gene family and function

Autophagy

Anti-apoptosis

Cell Cycle

Apoptosis Signaling

Nuclear Receptor

Senescense

Translation Regulator

DNA Repair

GPCR Signaling

Transmembrane Receptor

Transporter

Transcription Regulator

Enzymes

Delivery and viral packaging

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100

80

60

40

20

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rmal

ized

Lu

cife

rase

Exp

ress

ion

HEK-293

�Express-In �Lipofectamine 2000® �ExGen 500 �FuGENE 6®

COS-1 CHO C2C12 HepG2 Jurkat

Superior transfection efficiency seen with Express-In transfection reagent compared* to commercially available transfection reagents in various cell lines.

Packaging Mix

TransferVector

Transfection

Transcription

Packaging Cell

Viral Proteins

Assembly& Budding

Harvest ViralSupernatant

ReplicationIncompetent

VirusTransduce Cells

Titer 1-5x106 TU/mL

RNA

Maximize delivery with Thermo Scientific Open Biosystems Express-In transfection reagent

Express-In transfection reagent is a proprietary lipopolymeric formulation developed and optimized for transfection of plasmid DNA into the nucleus of cultured eukaryotic cells. Polymers, not cationic lipids, protect DNA in the cytoplasm and promote entry into the nucleus of transfected cells11.

• Polymeric formulation maximizes delivery into the nucleus

• High efficiency delivery to adherent and suspension cell lines

• Minimal cytotoxicity

Thermo Scientific Open Biosystems Trans-Lentiviral packaging system for high efficiency and biosafety

The most effective strategy for efficiently expressing your gene of interest.

VERSATILE: Effective packaging of second or third generation lentiviral vectors

UNSURPASSED BIOSAFETY: Genes encoding the components required for packaging the viral genome are separated across five plasmids minimizing the threat of recombinant replication competent virus production

BROAD TROPISM: Generate lentiviral particles that effectively transduce both dividing and non-dividing mammalian cells in vitro and in vivo

HIGH-TITERS: Generate titers of 1-5 x 106 TU/mL; virus can be further concentrated up to 1 x 109 TU/mL

www.thermo.com/GeneExpression 10

* Comparison experiments were performed using manufac- turers recommended protocols.

Use our gene-based search to find the right product for your research

Gene search and Online BLAST: www.thermo.com/openbiosystems

1. What is your gene of interest? Type the official symbol or accession related to your gene of interest into the gene search that is located at the top of every webpage.

To quickly find Incyte Clones, ESTs and clone homologs from different species, BLAST your sequence of interest against all 11 million sequences in our clone collection.

2. Search returns the hits organized by product tab. The blue bar under each tab shows the number of clones returned for each.

3. Click the product tab you are interested in to view the specific clones for each. Genbank accession and a brief description of the clone, species and vector are available for each clone. Clicking on Clone ID brings up additional clone details including sequence.

4. If additional filtering is required then click ‘Change Filters’ to narrow the results by species or vector type. The number of clones shown in each tab’s count will decrease accordingly.

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5. Add a clone to your cart by clicking on the cart icon. A check mark will appear when it has been added.

References 1. MGC (Mammalian Gene Collection) Program Team (2002) Generation and Initial Analysis of more than 15,000 Full-Length Human and Mouse cDNA Sequences. PNAS 2002 99:26; 16899-16903

2. MGC Project Team (2004) The Status, Quality, and Expansion of the NIH Full-length cDNA Project: The

Mammalian Gene Collection (MGC). Genome Res. 2004: 14; 2121-2127

3. MGC Project Team (2009) The Completion of the Mammalian Gene Collection (MGC). Genome Res. 2009: 19; 2324-2333

4. Jason M. Johnson, et al. (2003) Genome-Wide Survey of Human Alternative

Pre-mRNA Splicing with Exon Junction Microarrays. Science 302; 2141

5. Ostler, KR, et al. (2007) Cancer Cells Express Aberrant DNMT3B Transcripts Encoding Truncated Proteins. Oncogene 26: 38; 5553-5563

6. Rual, JR et al. (2004) Human ORFeome Version 1.1: A Platform for

Reverse Proteomics. Genome Research 14, 2128-2135

7. Lamesch et al. (2007) hORFeome 3.1: A Resource of Human Open Reading Frames Covering Over 10,000 human Genes. Genomics 89:307-15.

8. Rual et al. (2005) Towards a Proteome-scale

Map of the Human Protein-protein Interaction Network. Nature 437:1173-8.

9. Hopkins A.L. (2006) Nature Reviews Drug Discovery 5:993-996.

10.Sakharkar K.R. (2007) Current Drug Discovery Technologies 4: 48-58.

11.Pollard et al. (1998) Polyethylenimine but Not Cationic Lipids Promotes Transgene Delivery to the Nucleus in Mammalian Cells. The Journal of Biological Chemistry, 273, 7507-7511.

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Thermo Scientific Open Biosystems cDNAs and ORFs

Euro Literature # PB 2010 45

US Literature # 00025232B01U

Contact Information

Thermo Scientific Gene Expression Products

North America601 Genome Way, Suite 2100Huntsville, AL 35806Toll free: 888-412-2225Tel: 256-704-4848Fax: 256-704-4849

www.thermo.com/openbiosystems

US e-mail: openbiosystems@thermofisher.com

Europe e-mail: techservice.emea.genomics@thermofisher.com

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Other Countries:Technical support: +44 1372 840410

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