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ORFPOOLWG Sigma-Aldrich

MISSION® TRC3 Human Whole Genome Lentiviral ORF Pool

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Properties

application(s)   capture ELISA: 5 × 108 TU/mL using p24
shipped in   dry ice
storage temp.   −70°C

Description

General description

Sigma-Aldrich is proud to offer the largest collection of fully sequenced and pre-cloned human open reading frames (ORFs) to-date, available in a pooled lentiviral whole genome format. Developed at the Broad Institute, the MISSION® TRC3 human whole genome pooled ORF library consists of over 17,000 unique ORF clones, encoding for over 14,000 genes used for overexpression screening. This exclusive library contains a unique 24 nucleotide barcode sequence associated with each ORF clone for easy hit identification and deep sequencing/deconvolution.
This ready-to-use MISSION TRC3 Human LentiORF pooled library allow for stable integration, enrichment of cells, and long-term gene expression in difficult-to-transfect cell lines utilizing our best-in-class lentiviral manufacturing. This collection provides researchers with the unique tools to gain insights into gene function through modulating gene and protein expression.

The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. The whole genome lentiviral ORF pool fills this gap and provides researchers with a powerful research tool for carrying out crucial biological experiments.

Whether looking to perform gene overexpression studies or validate gene knockdown/knockout results from an RNAi or CRISPR experiment, LentiORFs are the ideal shortcut to protein expression and tool for gene analysis. Researchers also need to validate “hits” from a screen to confirm to eliminate false positives. This means confirming whether the observed phenotype is due the target gene of interest. Re-expressing the gene of interest via lentiORFs is the most accepted way to validate a gene target.

MISSION TRC3 Human LentiORF pooled whole genome library is provided in 8×25 μl aliquots at a minimum titer of 5×108 TU/ml (measured by a p24 assay).

Application

Functional Genomics/Overexpression Screening/Target Validation

Features and Benefits

• Efficiently screen the whole human genome (14,000+ genes) at the bench-top without robotics or specialized equipment
• Pre-cloned, sequence confirmed, expression-ready clones
• Ideal Shortcut to Protein Expression: Use Pooled Lentiviral ORFs to express protein-coding genes to assess their function
• Simple Deconvolution: The puromycin library is barcoded, consisting of a 24 nucleotide sequence located downstream of the V5 tag and translation stop. Each clone contains a unique barcode sequence that can be easily identified, allowing for simple deconvolution of pooled screening experiments
• EF1a promoter drives expression of the ORF construct, offering an alternative to the CMV promoter which may not be appropriate in certain cell types
• Puromycin resistance allows for double selection experiments in cells containing Neomycin or Blasticidin resistance
• Enriched population of cells: Use puromycin selection after transduction, for creation of a long term-stable cell line
• V5 epitope tag facilities pull-down and co-immunoprecipitation experiments to determine protein-protein interactions or label and visualize ORFs via immunofluorescence
• Positive and negative selection controls (BFP and GFP) are provided, which allow researchers to confidently gauge the success of their pooled screening experiments
• Lentiviral ORFs can infect a broad variety of mammalian cells (including difficult to transfect or primary cell lines) via transduction to facilitate gene overexpression for screening applications
• Rapid and affordable screening with easy-hit identification via Sigma-Aldrich′s deconvolution service
• Save time: Skip PCR, cloning, and DNA sequence verification steps

Preparation Note

Puro Kill Curve and Determining CFU (Colony Formation Unit) per mL. Prior to performing a library-scale screening, two preliminary experiments must be conducted. Visit sigma.com/lentipools or refer to the technical bulletin.

Other Notes

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. Though the lentiviral transduction particles produced are replication incompetent, it is recommended that they be treated as Risk Group Level 2 (RGL-2) organisms in laboratory handling. Follow all published RGL-2 guidelines for laboratory handling and waste decontamination.

Legal Information

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

Safety & Documentation

Safety Information

RIDADR 
UN 3245 9

Documents

Certificate of Analysis

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Protocols & Articles

Articles

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...
Keywords: Cancer, Cloning, Culture media, Functional genomics, Gene expression, Gene knockdown, Genetic, Genomics, Polymerase chain reaction, Proteomics, Purification, Recombination, Transcription, Transduction

Safe Handling of Lentivirus

Our lentiviral vector systems have been developed with enhanced safety features. Numerous precautions are in place in the design of our lentiviruses to prevent replication, including eliminating viru...
Keywords: Environmental, Genetic, Recombination, Transfection

Protocols

CRISPR Lentiviral Screening: Antibiotic Selection

A kill curve is a dose-response experiment where cells are subjected to a range of antibiotic concentrations to determine the minimum concentration of antibiotic needed to kill all cells over a desir...
Keywords: Antibiotics, Apoptosis, Gene expression, Transduction

Protocol, FACS Titration of Lentivirus

FACS Analysis of CFU FACS (Fluorescence-Activated Cell Sorting) provides a method for sorting a mixed population of cells into two or more groups, one cell at a time, based on the specific light scat...
Keywords: Enzyme-linked immunosorbent assay, Flow cytometry, Gene expression, Titrations, Transduction

Successful Transduction Using Lentivirus

Successful targeting relies on optimizing key sensitive steps in the process, including lentiviral transduction. Below are some helpful handling and titration tips from our R&D lentiviral experts. As...
Keywords: Antibiotics, Enzyme-linked immunosorbent assay, Gene expression, PAGE, Polymerase chain reaction, Titrations, Transduction

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Technical Service:

Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others.

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