GFC Transfection Arrays™ for High-throughput Over-expression Screening for Functional Genes in a Biological Process

Unlike siRNA screening library, which has been widely adopted by researchers to identify genes in a cellular phenotype through down-regulation, GFC Transfection Arrays provide a different approach to screen human genes for cellular phenotypes by gene overexpression. No longer must gene overexpression experiments be done one gene at a time. This white paper describes how GFC Transfection Arrays work and a variety of ways in which overexpression arrays have been used.

Overexpression screening technology, first developed for GFC Transfection Arrays in 2004, has been validated by peer-reviewed publications and is suitable for a wide range of biological studies and automated readout assays. The format for a GFC Transfection Array using full-length human cDNAs (OriGene TrueClones™) is a single-use 384-well plate, each well containing a distinct expression-ready full-length cDNA. GFC Transfection Arrays are economical (as low as $2 per gene), so that systems-biology approaches to study subsets of the human genome (transcription factors, for instance) are feasible for any laboratory. At the same time, GFC Transfection Arrays can be customized for the needs of bio-pharmaceutical companies devoted to genome-wide high-throughput screening.

Thus, GFC Transfection Array overexpression screening complements downregulation screening based on siRNA. One might say GFC Transfection Arrays provide the other half of the story.

GFC Transfection Arrays Maximize Convenience

A typical GFC Transfection Array overexpression screening experiment proceeds in four steps, as illustrated by the adjacent figure.

First, open the sealed GFC Transfection Array 384-well plate, where normalized human cDNA clones for overexpression screening are already distributed for transfection. Every plate includes two vacant columns that are included for the flexible arrangement of the researchers control samples.

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

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

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

Reverse Transfection. GFC Transfection Arrays are designed to allow use of the reverse transfection method for high-throughput overexpression as an assay format. 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.

Stable Platform. A notable aspect of GFC Transfection Arrays is the use of lyophilized DNA which makes long term storage possible. OriGene carefully lyophilizes cDNAs to be transfected to the plate during plate manufacture and then seals the plate for storage. Transfection is appropriate for most cell types when used with low-toxicity transfection reagents.

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

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

• Full-length cDNAs. Each well contains verified full-length cDNA plasmid
• No Subcloning. Each cDNA is subcloned into the same expression vector, pCMV6; the vector’s Cytomegalovirus promotor drives high expression in virtually all mammalian tissues
• Preloaded Plasmids. Every GFC Transfection Array 384-well plate comes with cDNA expression plasmids individually 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 is designed with empty wells for addition of user supplied positive and negative controls
• Data in Triplicate. OriGene supplies GFC Transfection Array plates in triplicate so there are three replicate assay data points per cDNA

GFC Transfection Array Versatility

GFC Transfection Arrays can be used to study a wide range of biological processes and are provided in many different human gene subsets. GFC Transfection Arrays are also easily adapted to many different readout assays.

Biological Processes. Processes that have been studied with overexpression assays include:

• Cell growth
• Cell differentiation
• Apoptosis
• Tissue-specific gene expression
• Developmental control

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 this problem is to offer GFC Transfection Arrays tailored to the study of specific human gene subsets for which there is wide research interest. Because these subsets contain hundreds of cDNAs rather than thousands, high-throughput screening costs drop drastically and become affordable for any laboratory.

Assay Readouts. GFC Transfection Arrays fit most plate readers for assay readouts that include:

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

Conclusion

One of the greatest frustrations in research is the time it takes to prepare experimental materials. So once a new time-saving technology becomes validated in the scientific literature, it is entirely predictable that the technology will become commercially available in a convenient, cost-effective form.

This has now happened for high-throughput gene overexpression for human functional genomics and the breadth of OriGene’s TrueClone content makes this possible. GFC Transfection Arrays make timesaving gene overexpression screening available and affordable for complementing gene downregulation screening with siRNA. The day of doing gene overexpression experiments one at a time—thanks to GFC Transfection Arrays—is over.

References

1. Huang Q, et al., (2004) Identification of p53 regulators by genome-wide functional analysis. Proc Natl Acad Sci USA. 101: 3456-61.
2. 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.
3. 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 Transfection Array human cDNA subsets, visit:
http://www.origene.com/cdna/GFC Transfection Array/default.mspx

To learn about other overexpression array experiments and assay readouts, 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 30,000 full-length human cDNAs, visit:
http://www.origene.com/cdna

Learn about the full range of OriGene products by visiting:
http://www.origene.com

Notice
GFC Transfection Arrays are for research use only. Use for diagnostic or therapeutic purposes is strictly prohibited. Reverse engineering of individual cDNA clones is not allowed.