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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a.
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 [email protected] 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
120
100
80
60
40
20
0No
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: [email protected]
Europe e-mail: [email protected]
BelgiumTel: 0800 80543Fax: 0800 80178
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SwitzerlandTel: 08005 63505Fax: 08008 38669
Other Countries:Technical support: +44 1372 840410
© 2010 Thermo Fisher Scientific Inc. All rights reserved. Gateway and Lipofectamine 2000 are registered trademarks of Life Technologies Corporation. Fugene is a registered trademark of Fugent, LLC. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
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