MISSION® TRC3 Human ORF Collection

Our ready-to-use MISSION® TRC3 Open Reading Frame (ORF) clones allow for stable integration, enrichment of cells, and long-term gene expression in difficult-to-transfect cell lines utilizing our best-in-class manufacturing.  This collection provides researchers with the unique tools to gain insights into gene function through modulating gene and protein expression. The MISSION® TRC3 ORF collection consists of over 33,000 pre-cloned ORFs, spanning more than 14,000 human genes.

Whether looking to perform gene overexpression studies or validate gene knockdown/knockout results from your RNAi/CRISPR experiments, TRC3 ORFs are your ideal shortcut to protein expression and gene analysis. Researchers often need to verify “hits” from a genetic screen in order to eliminate false positives caused by off-targets.  Validation confirms that the observed phenotype is due to knockdown/knockout of the target gene of interest. Re-expressing the gene of interest through ORF overexpression is the best and most accepted way to identify whether the phenotype change is the result of a true hit or is caused by off-target effects.

What is an ORF?

An open reading frame (ORF) is a sequence of DNA beginning with an initiation methionine codon, ATG, and ending with one of the three termination codons, TAA, TAG or TGA. This sequence is transcribed and translated into a message RNA and protein, respectively. While the genomic sequence of an encoded gene includes both introns (untranslated) and exons (translated) sequence, as well as 5’ and 3’ untranslated regulatory regions (UTRs), an ORF only encompasses the codon sequence of a translated gene. An ORF is also often known as the CoDing Sequence (CDS), as it only includes the translated codons and no regulatory sequence. ORFS are often inserted into expression plasmids (containing a promoter) and transduced into cells for the study of the corresponding gene and protein function. ORFs also play an important role in RNAi and CRISPR rescue experiments, where gene expression is restored (gain-of-function) after gene ablation for phenotypic validation.

MISSION® TRC3 Human ORF Collection

MISSION® TRC3 Human ORF collection includes a comprehensive collection of fully sequenced ORFs that are packaged in 2 different Gateway-adapted vectors: Entry vector and Puromycin vector.  All Entry vector clones are available in bacteria glycerol stock and plasmid DNA format.  All Puromycin vector clones are available in bacteria glycerol stock, plasmid DNA, and lentiviral particle format.

We offer over 33,000 pre-cloned human ORF constructs targeting more than 14,000 human genes that may be used for overexpression or rescue/validation experiments. Our collection contains the exclusive puromycin library and the expanded Human ORFeome v8.1 Entry library  (Yang et al 2011).

Our ORF expression vector system allows for additional peptide sequences or tags to modify properties of the native protein. Affinity tags can be used to purify the protein for further structural and characterization studies. Epitope tags (such as V5 found in the puromycin vectors, or a tag of your choice when subcloning from the entry vector) allow for simple detection or purification via antibodies. The addition of reporters and fluorescent tags also allows for gene expression monitoring and cellular localization.

 

The Gateway® cloning system

Figure 1. The Gateway® cloning system (Invitrogen) allows the transfer of DNA fragments between plasmids containing the ‘Gateway att’ flanking sites. A ‘Gateway entry clone’ is prepared by inserting the DNA fragment/gene of interest in between ‘att’ flanking repeats in a plasmid.

The gene of interest from the Gateway Entry clone can be transferred to any Gateway Destination vector (that contains ‘att’ sticky ends, promoter, epitope tag, selection marker) to produce an ‘expression clone’ for further applications.

The Human ORFeome v8.1 library (Entry Library)

  • The Entry library is created by transferring fully sequenced ORFs from Mammalian Gene Collection (MGC) cDNAs into a recombinational entry vector pDONR223 by adapting the Gateway cloning system.
  • The library is used in applications where rapid ORF shuttling into any Gateway®-compatible expression vector is required.
  • The native stop codon is omitted and replaced with one of the three termination codons, TAA, TAG or TGA
  • There is no upstream promoter so it is not to be used for expression studies (unless subcloned into an expression vector).
  • TRC3 Entry collection Includes expanded gene and clone content from the hORFeome v8.1 library, boosting coverage up to 14,000 human genes (16,000+ clones).
  • Easily and efficiently transfer to any Gateway-adapted expression vector containing different promoters, selection markers, fluorophores, tet-inducibility, etc.
  • The library is not available in lentivirus format, as it lacks the necessary lentiviral components for viral packaging. It is available in bacteria glycerol stock and plasmid DNA formats.

pDONR223 vector map and features table

Figure 2. pDONR223 vector map and features table
 

Vector Label Function
att Gateway recombination site
ORF Open reading frame insert
pUC ori High copy replication and maintenance in E. coli
Spcr Spectinomycin resistance gene

 

Puromycin ORF Library

  • The library consists of a collection of 17,000+ human ORFs.
  • The ORFs are pre-cloned and ready-to-use for mammalian cell expression.
  • The Expression-ready vectors have an EF1-alpha promoter driving ORF expression that is preferred for some cell types (i.e. primary cells) where the CMV promoter gets turned off or methylated.
  • 5’ V5 epitope allows for protein purification or coprecipitation studies.
  • PAC (puromycin resistance gene) acts as a selective marker.
  • The ORF library is exclusive to Sigma-Aldrich
  • Pooled screening applications: Contains a 24 nucleotide barcode sequence located downstream of the V5 tag and translation stop. Each clone’s unique barcode sequence can be found in the library inventory file for easy identification upon deconvolution.
  • The library is available in bacteria glycerol stock, plasmid DNA, and lentiviral particle formats.
  • The ORF constructs can be transfected as plasmids or transduced as lentiviral particles in difficult-to-transfect cells.
  • The level of ORF expression can be regulated by controlling the ratio of functional viral particles to cells.

 

TRC3 Puromycin vector map and features

 

Figure 3. TRC3 Puromycin vector map and features
 

Vector label Function
5’ LTR Chimeric 5' long terminal repeat
Ψ Psi viral genome packaging signal
RRE Rev Response Element - increases full-length viral packaging
SV40 Simian virus 40 (SV40) promoter
PAC Puromycin N-acetyl-transferase (PAC) gene confers puromycin resistance
hEF1α RNA Polymerase II Promoter Human Elongation Factor-1 alpha
ORF Open reading frame insert
v5 V5 epitope tag
Stop One of the three termination codons: TAA, TAG or TGA
WPRE Woodchuck Posttranscriptional Regulatory Element: enhances transcription of transgenes
3’ SIN LTR 3' Self-inactivating long terminal repeat
F1 ori Phage-derived ORI that allows for the replication and packaging of ssDNA into phage particles
Ampr Ampicillin bacterial selection marker
pUC ori High copy replication and maintenance in E. coli
Barcode (not shown) Pooled screening application barcode

 

The MISSION® TRC3 Advantage

  • Value in validation: Perform RNAi or CRISPR rescue experiments after screening to validate gene hits.
  • Lasting results: Lentiviral based system produces constitutive expression of ORFs to create long-term, stably expressing, and enriched cell populations. Lentiviral vectors are ideal for use in difficult-to-transfect or primary cell lines.
  • Screen with confidence: Exclusive TRC3 Puromycin ORF library uses cost-effective pooled screening capabilities for high-throughput, large-scale screening capabilities and expression of thousands of ORFs.
  • Save time: Skip PCR, cloning, and DNA sequence verification steps.

Although cDNA synthesis is commonly performed in research labs, it can be very costly in terms of time and money. Creation of your own clones is error prone and can take several steps over many days, and require sequence verification (Figure 4 below). Additionally, mutations may be introduced, and experiments may have to be repeated to get the desired transcript.

Our fully sequenced ORF clones are an ideal shortcut to protein expression. Pre-cloned MISSION® TRC3 ORFs eliminate the uncertainty of de novo cloning and our online ordering tool allows you to search for ORF clones and select the best match for your gene(s) of interest.

Traditional cDNA synthesis

Figure 4. Traditional cDNA synthesis

 

MISSION® TRC3 ORF Formats

MISSION® TRC3 ORFs are available in bacterial glycerol stock, purified DNA or viral particle formats. Choose from whole genome arrayed or pooled libraries, custom gene panel sets, or individual ORF clones. The MISSION® TRC3 Collection is customizable and accommodates a wide variety of research needs.

Bacterial glycerol stock format:

  • E. coli cells have been transformed with TRC3 ORF plasmids and cell stocks are supplied in media containing 10% glycerol as a cryopreservative.
  • Bacterial glycerol stock is a renewable resource - simply inoculate fresh culture media and grow overnight to generate fresh culture.
  • The plasmid must first be purified from a fresh bacterial culture in order to begin gene expression and validation experiments.
  • TRC3 Entry plasmids containing the ORF of interest must be subcloned into a compatible vector via Gateway cloning prior to use in mammalian cells.
  • MISSION® TRC3 Entry and Puromycin collections are available in bacterial glycerol stock format.

Plasmid DNA format

  • DNA is supplied as purified plasmids that are ready for subcloning or expression
  • TRC3 Puromycin plasmids containing the ORF of interest can be used to transfect cells or can be packaged into replication-incompetent lentivirus for transduction into desired cell cultures or in vivo.
  • TRC3 Entry plasmids containing the ORF of interest must be subcloned into a compatible vector via Gateway cloning prior to use in mammalian cells.
  • MISSION® TRC3 Entry and Puromycin collections are available in plasmid DNA format.

Viral particle format:

  • Contains the TRC3 ORF plasmids packaged into lentivirus for transduction into desired cell lines and in vivo.
  • High titer and high volume options are available (105-109 particles/mL, 0.1mL -10mL).
  105 particles/mL 106 particles/mL 107 particles/mL 108 particles/mL 109 particles/mL
0.1mL    
0.2mL
1 mL
2 mL
5 mL  
10 mL  
  • MISSION® TRC3 Puromycin collection is available in viral particle format. The Entry collection is not available in viral particles since the vector lacks the necessary viral elements for packaging virus particles.

Upon viral transduction, the ORF expression plasmid DNA integrates into the DNA of the host cell

Figure 5. Upon viral transduction, the ORF expression plasmid DNA integrates into the DNA of the host cell. The cellular machinery recognizes the promoter and synthesizes the protein. The resulting gene product and translated protein can then be used for purification, localization for characterization of protein function, or to determine if the altered phenotype is a result of gene overexpression.

How are the ORFs classified?

ORF clones are classified on stringency to the corresponding NCBI Refseq transcript.

Perfect Match: the ORF matches 100% in both nucleotide and protein sequence.  

Near-Perfect Match:  the ORF has at least a 99% protein match (if coding) or at least a 99% nucleotide match (if non-coding).

Lower Match:  the ORF has a good "partial" match, but may contain large contiguous indels indicating e.g. truncation or missing/extra exon.  Score is calculated using protein match (if coding) or nucleotide match (if non-coding).

Gene Rescue/Validation

RNAi Rescue

RNA interference assesses the functionality of human genes that mediate cellular phenotypes by conducting loss-of-function gene studies. In addition to gene-specific silencing through perfect complementarity to the target transcript, RNAi can cause phenotypic changes by the regulation of unintended transcripts through seed-region complementarity that can lead to false interpretation of target gene function.

It is necessary to validate target specificity of siRNA or shRNA-mediated phenotypes via RNAi rescue experiments. A common method of phenotypic rescue involves the use of exogenously expressed transcripts that are resistant to siRNA or shRNA targeting.

These transcripts are obtained from the TRC3 Human ORF collection.  TRC3 ORFs lack the 3’UTR and 5’UTR, rendering the ORFs resistant to siRNA or shRNAs that target the gene transcript’s 3’UTR. The siRNA/shRNA will silence only the endogenous expression of the gene target that contains 3’ or 5’UTRs. If a siRNA/shRNA target lies within the coding sequence of the ORF, site-directed mutagenesis can be easily completed using commercially available kits to introduce silent mutations into the binding region, rendering the mutated ORF resistant to inhibition.

If exogenous expression of the ORF for the gene of interest results in phenotype rescue, it confirms that the phenotype is due to target-specific effects of the shRNA/siRNA. If no phenotype rescue is observed, it indicates that the shRNA/siRNA effect may be non-specific or off-target.

shRNA Product Offering

siRNA Product Offering

CRISPR Rescue

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is an effective method to selectively knock out gene expression. It is a ribonucleoprotein (RNP) complex involving Cas9 protein and guide RNA (gRNA). gRNA targets the genomic sequence of interest and directs the Cas9 nuclease to create double-stranded breaks in the DNA. Gene knockout is the result of frameshift mutations caused by insertions or deletions at the break site that naturally occur in the process of  DNA repair via Non-Homologous End Joining (NHEJ).  Gene knockout is permanent and heritable (Marraffini & Sontheimer, 2010).

As with RNAi studies, it is important to rescue a CRISPR/Cas9 knockout experiment to ensure that the observed phenotype is specific to the intended target. There are many bioinformatic platforms available to interrogate the off-target effects of CRISPR/Cas9 guide RNAs, but the gold standard experiment will always be to introduce a CRISPR/Cas9 resistant ORF and re-evaluate the phenotype. If the exogenous ORF restores wild-type function, then the effect can be attributed to the gene. If the phenotype is not reversed, then off-target effects of the guide may be responsible. Similar to the technique outlined above, the exogenous ORF can be mutated at the PAM site to render it resistant to CRISPR/Cas9 cleavage. In addition, it has been shown that guide RNAs which target the 3’ end of a coding exon and extend into the 5’ intron will not target an exogenous ORF since ORFs do not contain introns. This technique can be used without modification of the ORF if your CRISPR/Cas9 strategy allows.

From (Incontro, 2014):

[... it is essential to perform rescue experiments that definitively rule out off-target effects as the cause of the observed phenotype. For the rescue, it is important to ensure that the rescue construct will not itself be targeted by CRISPR... We searched for a specific gRNA sequence encompassing an intron-exon junction such that the cleavage site (position 3) would be in the exon, but most of the gRNA sequence would lie within the intron. By using this very simple strategy, it is now possible to perform rescue experiments using unmodified cDNA, which greatly simplifies and accelerates the validation of the phenotype."

Gene Overexpression Studies

While RNAi and CRISPR technologies allow scientists to genetically alter or silence targeted genes, ORFs are valuable tools that do more than simply confirm gene knockdown/knockout hits.  ORFS can be used independently of CRISPR or RNAi systems to “turn on” or “overexpress” particular genes. Flipping the switches on genes one at a time can help reveal the functions of individual genes, such as those that play a role in cancer. ORFs have been used in several studies where gene screening, expression analysis and genetic rescue are important. Over-expression of genes and proteins is widely used in functional genomics, proteomics and system biology studies.

Some applications of gene overexpression studies

Figure 6. Some applications of gene overexpression studies. Adapted from: Gene Overexpression: Uses, Mechanisms, and Interpretation (Prelich, 2012)

Some of the studies include:

  1. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation
  2. KRAS and YAP1 converge to regulate EMT and tumor survival

CONTACT US

For questions or inquiries, please email MISSIONRNAI@sial.com

MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.

Materials

     

 Works Cited

  • Eisenstadt, L. (2010, November 30). What is an ORF? Retrieved from https://www.broadinstitute.org/blog/what-orf.
  • Incontro S, e. a. (2014). Efficient, complete deletion of synaptic proteins using CRISPR. Neuron, 1051-7.
  • Marraffini, L., & Sontheimer, E. (2010). Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature, 568-71.
  • Prelich, G. (2012). Gene Overexpression: Uses, Mechanisms, and Interpretation. Genetics, 190(3), 841-854. doi:10.1534/genetics.111.136911.
  • Yang X, Boehm JS, Yang X, et al. A public genome-scale lentiviral expression library of human ORFs. Nature methods. 2011;8(8):659-661. doi:10.1038/nmeth.1638.