GFC-Arrays: quick and reliable screening for regulator genes via high-throughput functional perturbation

Introduction

Discovery of the key genes regulating a cellular process, a disease progression or a biological pathway has always been a major interest for most biomedical researchers. For decades, scientists have relied heavily on two main approaches. Some search for targets by transfecting entire expression libraries into appropriate cells to screen for genes that produce a desired readout via functional complementation. Due to high library complexity and low target gene transcript abundance, this process is laborious and insensitive. The second approach is to functionally test individual candidate genes one by one. This is an extremely slow process requiring years of data collection, and the chance for ultimate success is low. Identifying and validating novel targets has become an increasingly difficult task, and the research community awaits innovative technologies to advance the discovery process.

OriGene’s GFC-Array (Genome Wide Full-length cDNA Arrays) is a revolutionary tool that allows scientists to break through the technical barriers associated with traditional target identification. It enables quick, sensitive and reliable identification of key genes in a cellular process of interest. As the world’s largest source of full-length human cDNA clones, OriGene is in the unique position to convert its TrueClone Collection, a collection containing over 24,000 expression-ready cDNAs, into functional screening cDNA arrays. In a GFC array, hundreds or thousands of individual full-length cDNAs are arrayed into a multi-well format. Each well contains 60 ng of a single expression-ready cDNA, a concentration optimized for transfection into a variety of cells used in functional activity assays. A researcher can simply add cells to the GFC plate and apply his/her reporter assays. Once a well shows a distinct readout, the gene in that well can be immediately identified as the underlying cause for the functional perturbation.

Similar to siRNA libraries, GFC-Arrays provide individual gene perturbing capability in a high-throughput format. Unlike siRNA libraries which down-regulate gene expression, GFC-Arrays over-express individual gene expression. Compared to the knock-down system, the up-regulation system is generally more robust. Furthermore, GFC arrays and siRNA screening libraries are complementary and can be used in tandem to validate the collective results. This white paper describes how GFC-Arrays work and the variety of ways in which over-expression arrays have been used.

Multiple leading pharmaceutical and academic researchers have provided the proof of principal for GFC-Arrays. Most prominently, in 2005, scientists at the Genomics Institute of the Novartis Research Foundation successfully used 20,000 expression-ready cDNAs to identify the known and novel regulators in the Wnt signal pathway(1). Several publications describing similar technologies have further demonstrated the validity, simplicity, versatility and usefulness of this technology (2-5).

OriGene has engineered its GFC arrays to be suitable for a wide range of biological studies and automated assay readouts. The format for a GFC-Array using full-length human cDNAs (OriGene TrueClones™) is a disposable 384-well plate. These plates are expressly produced to minimize the amount of work required in preparing experimental materials. GFC-Arrays are economical (as low as $2 per gene), so that systems biology approaches to study subsets of the human genome (transcription factors, for example) become feasible for any laboratory. At the same time, GFC-Arrays can be customized for the needs of biopharmaceutical companies devoted to genome-wide high-throughput screening.



GFC-Arrays Maximize Convenience

A typical GFC-Array over-expression screening experiment proceeds in four steps, as illustrated by the adjacent figure.

First, open the sealed GFC-Array 384-well plate, where human cDNAs for over-expression screening are already distributed for transfection. Every plate includes two vacant columns that are included for the flexible arrangement of the researcher’s control samples.

Second, if the readout assay will use a reporter gene such as a promoter-driven luciferase, add the reporter plasmid along with a transfection reagent. The users own controls are then added to the empty columns.

Third, add cells and incubate the plate for reverse-transfection.

Fourth, add assay reagents and use a plate reader to score the plates.

Reverse Transfection. A notable aspect of GFC-Arrays is its use of reverse transfection, which makes high-throughput overexpression possible. For reverse transfection, OriGene lyophilizes cDNAs to be transfected to the bottom of the plate during array preparation and then seals the plate for storage. OriGene has optimized the reverse transfection reaction with regard to DNA quantity, cell number and transfection reagent. The standard protocol is appropriate for most commonly used cell types. For specialty cells, the researcher must optimize the procedure; OriGene provides “Optimization Plates” employing reporter expression plasmids to facilitate such an effort.

Hit Validation. After screening with GFC-Arrays, the next step is usually to validate hits. It is important to verify that an observed phenotype is truly due to cDNA overexpression and is not an experimental artifact. We recommend validating each hit via standard transfection with the specific cDNA plasmid. All cDNAs in GFC-Arrays are available separately in 1µg to 10 µg amounts.

Minimal User Requirements. A true high-throughput screening system minimizes the time and effort needed to obtain accurate, reproducible data. GFC-Arrays limit user-supplied materials to the following:

• A cell line for DNA introduction
• A readout assay to score the GFC plates
• A 384-well plate reader
  
  

Standard Features. To maximize user convenience, these GFC-Array features are standard:

• Full-length cDNAs. Every well contains verified full-length cDNA
  
• Uniform expression. Each cDNA is cloned in the same expression vector, pCMV6;
  the vector’s Cytomegalovirus promoter drives high expression in virtually all tissues
  
• Pre-Normalized plating. Every GFC-Array 384-well plate comes with cDNA expression plasmids already distributed into the wells; 60 ng of each is standard
  
• Quick Transfection Optimization. An Optimization plate enables fast determination of optimal transfection conditions and reagent concentrations
  
• Standard Controls. Every plate comes with empty wells for customized in-plate positive and negative controls
  
• Data in Triplicate. OriGene supplies GFC-Array plates in triplicate so there are three
  data points for each cDNA
  
  

GFC-Array Versatility

GFC-Arrays can be used to study a wide range of biological processes and many different human gene subsets. GFC-Arrays are also easily adapted to many different assay readouts.

Biological Processes. Processes that have been studied with over-expression assays include:

• Cell growth
• Cell differentiation
• Apoptosis
• Tissue-specific gene expression
• Developmental control
• Cell sensitivity to small molecules
  
  

Human Gene Subsets. Sometimes the cost of screening OriGene’s entire TrueClone collection may be prohibitive or the effort to assay such a large number of cDNAs may not be practical. OriGene’s solution to these problems is to offer GFC-Arrays of gene subsets for which there is wide research interest. Because these subsets contain hundreds of cDNAs rather than thousands, high-throughput screening efforts and their associated costs are minimized and therefore become affordable for most laboratories.

Human gene subsets currently available for GFC-Array over-expression studies include:

• Transcription Factors
• Kinases
• Transmembrane Proteins
• Druggable Genes (genes for enzymes/receptors to which drugs can be targeted)
• Screening Sets
  
• More cDNA subsets are currently in development.
  
  

Readout Assays. GFC-Arrays are adaptable to plate readers for readout assays that include:

• Transcription reporter gene assays
• Receptor activation assays (e.g., G protein-coupled receptor assays)
• Nuclear translocations of protein markers
• Enzyme assays
• Immunoassays
• Protein interaction assays
• Cell proliferation assays
• Apoptosis assays
• Cell toxicity assays
• Single cell morphology assays

Overexpression Array Experiments from the Scientific Literature

High-throughput genome-wide overexpression assays for functional screening have been validated in many recently published papers. Here are three examples.

Screening for modulators of a transcription factor.

Huang Q, et al., (2004) Identification of p53 regulators by genome-wide functional analysis. Proc Natl Acad Sci USA. 101:3456-61.

In this study, 20,000 cDNAs were arrayed into 384-well plates in order to screen for proteins modulating the transcriptional activity of the p53 tumor-suppressor protein (2). The cDNAs were then introduced into human colon cancer HCT116 cells via high-throughput reverse transfection. Each transfection also introduced a reporter plasmid containing a luciferase gene controlled by a p53-responsive transcription element. A plate reader was used to read each well’s luciferase luminescence.

This genome-wide cDNA transient overexpression screen identified two known p53 activators and nine proteins whose ability to modulate p53 activity were previously unknown. Seven of the genes up-regulated p53 activity and two down-regulated p53 activity. Follow-up phenotype characterization in zebrafish, chick embryos, and mouse embryonic fibroblasts revealed that some of the nine proteins modified transcription of the p53 gene and others modified p53 activity at the post-translation level.

Screening for genes that ameliorate oxidative-stress toxicity.

Zitzler J, et al., (2004) High-throughput functional genomics identifies genes that ameliorate
toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide. Mol Cell Proteomics. 3:834-40.

Neuronal damage caused by reactive oxygen species (ROS) is associated with several neurodegenerative disorders. In this study, 5000 human cDNAs were arrayed into 96-well plates and then transfected into a mouse hippocampus cell line (3). The screening objective was to identify genes capable of counteracting ROS damage. To model oxidative stress-induced toxicity, after transfection the cells were challenged with hydrogen peroxide. The following day, the cells received a metabolic dye for detecting cell survival and proliferation. The automated readout assay scored cell viability by measuring dye fluorescence, which was directly dependent upon the level of hydrogen peroxide toxicity.

The experiment identified six genes for known antioxidative enzymes, and one gene for a transcription factor that regulates genes for oxidative-stress protection. The screen also revealed (and follow-up experiments confirmed) that a neuronal gene called GFPT2 (glutamine-fructose-6-phosphate transaminase 2) also enabled cells to survive hydrogen peroxide toxicity. This GFPT2 phenotype was previously unsuspected.

Screening for tumor suppressor phenotypes.

Koenig-Hoffmann K, et al., (2005) High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes. Int J Cancer 113: 434-9.

In this study, researchers transfected almost 600,000 independent cDNA library clones into HEK293 cells in order to identify novel tumor suppressor genes (4); maximum throughput was 40,000 transfections per 24 hours. The readout assay used colorimetric beta-galactosidase assays to detect transient expression of a lacZ gene controlled by a cAMP response element (CRE). As an indirect assay, the phenotype of interest was not high levels of beta-galactosidase but the loss of that signal, a possible indication of apoptosis induction or loss of cell viability, traits associated with tumor suppressor proteins.

Genome-wide screening detected several genes known to be tumor suppressors, indicating that the screening protocol was valid. Apoptosis associations were confirmed by further transfections in which cDNA-transfected cells were analyzed for apoptosis-specific events such as DNA fragmentation and caspase activation. In all, the experiment detected 89 genes previously unknown to be associated with apoptosis. Of them, 70 had not been previously associated with any phenotype.

Customized GFC-Arrays
For experiments with special requirements, OriGene offers customized GFC-Arrays. Customization options include, but are not limited to:

• Increasing or decreasing the cDNA amount per well
• Full-length human cDNA subsets specified by the client
• Special well coatings
• Special plates (including 96-well plates)

Customers are encouraged to contact us at Techsupport@origene.com or call 1-888-267-4436 to discuss your special request.

Special Promotions:

Currently OriGene offers two starter GFC-Arrays for mass screening of two major functional groups, transcription factors and protein kinases. At as low as $2/gene, the kits are affordable and allow quick data generation.

Transcription Factor GFC Array: Contains 704 commonly known transcription factors

Cat#: FTC10001

Kit contents:

• Three sets of 2 X 384-well plates
• One optimization plate for transfection condition control
• One copy of Application Guide
  

Price: $1500/kit

Protein Kinase GFC-Array: Contains 352 protein kinases

Cat#: FKI10001

Kit contents:

• Three sets of 1 X 384-well plates
• One copy of Application Guide
  Price: $ 1500/kit
  
  

Conclusion
GFC-Arrays combine the advantage of having a large number of verified full-length cDNAs in an uniform expression system with the convenience of high throughput reverse transfection. This new tool creates an unprecedented opportunity for researchers to interrogate hundreds or even thousands of individual genes for their functional involvement in a particular biological activity. OriGene supplies the transfection-ready cDNAs (grouped by functionalities) as pre-normalized plasmids in 384-well plates, allowing researchers to apply their own assay systems. Resulting gene hits can be immediately identified. For further investigation of all generated leads, microgram quantities of purified plasmids for the corresponding wells can be easily obtained from OriGene.

The GFC-Array is an innovative and validated tool that will greatly accelerate the process of target gene identification. The GFC-Array saves a tremendous amount of time for researchers normally dedicated to cDNA cloning, sequence verification, and plasmid preparation, and it allows the simultaneous screening of large numbers of genes. The GFC platform’s flexibility allows it to be successfully used in a diverse range of assays. OriGene’s GFC-Array, like siRNA screening libraries, will become more widely accepted and utilized as a general methodology in the biomedical discovery arena.


References

1. Liu, J, et. al., (2005) Identification of the Wnt signaling activator leucine-rich repeat in Flightless interaction protein 2 by a genome-wide functional analysis. Proc Natl Acad Sci USA. 102: 1927-32
2. Huang Q, et al., (2004) Identification of p53 regulators by genome-wide functional analysis. Proc Natl Acad Sci USA. 101: 3456-61.
3. Zitzler J, et al., (2004) High-throughput functional genomics identifies genes that meliorate toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide. Mol Cell Proteomics. 3: 834-40.
4. Koenig-Hoffmann K, et al., (2005) High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes. Int J Cancer 113: 434-39.
   
   

For More Information…
To learn more about available GFC-Array human cDNA subsets, visit:
http://www.origene.com/cdna/gfc-array/default.mspx
To obtain more references on over-expression array experiments and readout assays, see "Large Scale Functional Profiling Using cDNA Clone Collections" at:
http://www.origene.com/cdna/trueclone/systems_biology.mspx
To learn about OriGene’s collection of more than 24,000 full-length human cDNAs, visit:
http://www.origene.com/cdna
Learn about the full range of OriGene products by visiting:
http://www.origene.com