Feature: Automated genomics

This feature appeared in the May/June 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Read part I, Functional genomics writ large.

The Arrayed Retroviral Expression Cloning (ARVEC) facility’s new technology is automating the tracking of the differences caused by different genes, uncovering their functions.

The ARVEC centre purchased from Open Biosystems and Imagenes 17,100 open reading frames (ORFs) derived from the Mammalian Gene Collection, representing almost every currently available human gene.

Researchers used Invitrogen’s proprietary Gateway insertion technology to transfer the cDNAs into a lentivirus vector.

Commonly used retroviral vectors are only able to transduce actively dividing cells. ARVEC’s lentivirus vector has the ability to transduce non-dividing cell lines and integrate its packaged transgene into the host cell’s genome.

Dr Dubravka (Duka) Skalamera says this allows researchers to work with differentiated cells from biologically relevant tissues – both normal, and cancerous. A robotic microscope collects images of cells in multiple single colour channels in each well. Each colour tracks a different cell phenotype.

The blue fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI), binds to DNA of all cells and is used to image cell nuclei and determine cell cycle phase.

Green Fluorescent Protein (GFP) marks transduced cells and highlights details of cell size and shape, while the red fluorescent dye Cy5, attached to a nucleotide analogue, is incorporated only by cells synthesising new DNA marking proliferating cells only. Additional features, such as state of cytoskeleton, or cellular lipids can be tracked by other dyes or fluorescently labelled antibodies.

Skalamera says the process generates enormous volumes of data, hence the description: 'high-content imaging.' All the acquired data are then analysed in a bioinformatics pipeline.

“Researchers can now come to us with a particular disease – say, melanoma – in normal or tumorised cells, and compare the effects of knocking out different genes,” she says.

“We will work with them to develop an appropriate high-throughput assay, such as the proliferation assay described by ARVEC researcher Max Ranall in last year’s Biotechiques ,” Skalamera said. “Inactivating or over-expressing genes crucial to the development of melanoma will cause cells to proliferate when they are not supposed to, marking them as hits in a screen.”

The range of currently available human cell lines is the only constraint on the types of experiments that can be performed, but Skalamera says that, in principle, the ARVEC facility can do experiments for any disease that can be assayed in tissue culture.

Skalamera says the ARVEC facility’s location within the Diamantina Institute was the reason for its initial focus on cancer experiments, because many of the institute’s researcher, like Professor Tom Gonda, work on cancer.

The institute also has research expertise in immune system and bone disease and ARVEC is developing assays to identify genes involved in both. When crucial genes are identified they can be targeted by therapeutic agents.

“Because the Diamantina Intitute is located within a major hospital, the ARVEC facility is going to become part of a translational research institute. We have doctors who can take the results of our research to a more applied level.

“We’ve run a screen on normal non-tumorigenic breast tissue (soon to be published in PLoS One). The cell line requires an external source of certain growth factors, like Epidermal Growth Factor, to maintain normal growth and development.

“In a breast tumour, that requirement is bypassed – the tumour synthesises its own growth factors. We can experimentally over-express certain genes in normal breast tissue, and identify those that allow the cells to grow and divide in the absence of EGF.”

According to Skalamera, these are potential oncogenes and provide entry points for researchers to explore pathways that drive cell growth and proliferation, as well as targets for anticancer therapy.”

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Open invitation

When Diamantina Institute researchers Professor Tom Gonda and Dr Brian Gabrielli conceived the ARVEC facility, they set out to devise an automated high-throughput system that, in principle, would allow researchers to explore the function of any human gene capable of inducing a dominant phenotype in a biologically relevant cellular assay.

Gonda and Gabrielli wanted to create a world-leading commercial facility that, as well as servicing researchers at the Diamantina Instiute, could offer a commercial functional genomics screening service to any Australian or overseas researcher with a gene of interest – whether that involved determining its normal biological function, or assessing its oncogenic potential.

The Diamantina Institute researchers secured initial funding from the National Health and Medical Research Council, and the Australian Research Council to establish the facility.

They subsequently then obtained further funding from the Australian Cancer Research Foundation to purchase commercial human and mouse libraries of short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) for gene-knockout research, and for over-expression experiments, a library of 17,100 Open Reading Frames (ORFs) for every currently available human gene.

At the time of writing, the ARVEC facility was offering to perform a functional screening project, free, for an Australian researcher or research group with the most innovative screening proposal. The ARVEC team has invited inquiries for screening projects from all interested researchers.

This feature appeared in the May/June 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.