RNAi delivers knock-out punch

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

If you need a suspect oncogene silenced, who are you going to call? Whether you want a gene terminated, or merely taken out of the game for a while, who are you going to call?

Dr Kaylene Simpson, of the Peter MacCallum Cancer Centre in Melbourne, is the one to talk to. Simpson manages the new Victorian Centre for Functional Genomics (VCFG), which provides a ready-made RNA-induced gene-silencing service for Australian academic researchers.

RNA-induced gene silencing has become indispensable for exploring gene function, and is a powerful means of identifying genes involved in the development and spread of cancers.

But gene-silencing technology is expensive and the methodology demanding. The cost of running and staffing a large-scale, high-throughput facility is beyond the resources of most research institutions; others simply prefer to out-source gene-silencing and focus on its application – a common pattern as emerging technologies enter routine use.

In late 2006 Peter Mac molecular oncologist Associate Professor Ricky Johnstone saw the need for a centralised facility to provide a widely available gene-knockdown service to the Victorian medical research community.

With start-up funding provided by the Department of Innovation Industry and Regional Development (DIIRD), AMATA and Peter Mac, Johnstone and Peter Mac colleagues, including microarray facility manager Vikki Marshall, established the VCFG.

One of the first Australian researchers to apply large-scale RNAi to cancer genomics, Johnstone saw Peter Mac as a future technology leader in the field. He achieved a coup late last year by luring the Monash/La Trobe University-trained Simpson back from Harvard Medical School in Boston to manage the new centre.

In 2006, Simpson was a key member of the Walter and Eliza Hall Medical Research Institute research team, headed by Dr Jane Visvader and Dr Geoff Lindeman, that made international headlines by growing an entire, milk-secreting mammary gland from a single mouse mammary stem cell.

The VCFG is housed within the greater Australian Cancer Research Foundation (ACRF) Cancer Genomics Program, which also incorporates an Automated High Throughput siRNA Screening Facility, a Single Molecule DNA Sequencing Laboratory and the Peter Mac Microarray Facility.

The facility was established with joint funding from the Victorian State Government, the ACRF, Peter Mac, and technology support from the Australasian Microarray and Associated Technologies Association (AMATA). ---PB---

Open platforms

The functional genomics facility at Peter Mac is separated into two functional platforms. For longer-term gene knockdown tasks, the VCFG uses a lentivirus-delivered short hairpin RNAs (shRNA) system provided by Open BioSystems.

The ACRF is funding a complementary system that employs Dharmacon RNAi Technologies’ synthetic short interfering RNAs (siRNA) for transient gene knockdown.

Simpson says the integrated systems will form the largest and most comprehensive functional genomics facilities in Victoria. “The technology is open to all Australian academics on a cost-recovery basis, with costs kept to a minimum to ensure affordable access,” she says.

The lentivirus shRNA system incorporates a green fluorescent protein (GFP) marker that provides visible confirmation of insertion and expression of the hairpin RNAi construct. The construct also contains an antibiotic resistance gene that allows transformed cells to be selected by growing them in puromycin-laced media.

The VCFG provides whole-genome screening, using sub-libraries of shRNA constructs that contain 4608 different constructs. Simpson says that, on average, the whole genome covers two or three different sequences for each gene, to ensure efficient gene targeting.

“The siRNA platform works on a very different, gene-for-gene basis using robotic-driven approaches,” she says.

“For the shRNA screens, we produce the virus, and the researcher comes in and does the transductions. We help all our researchers with assay development, and provide standard operating protocols for all steps of the lentivirus infection process.

“We offer open access to researchers Australia-wide – for example, we already have researchers from the Garvan Institute in Sydney working with us – but currently, most come from Victorian research centres.”

Simpson says the centre is strongly focused on hunting and characterising genes involved in cancer, but its doors are open to the wider bioscience community.

“Our hairpin RNAi facility coves the entire human genome, but not the mouse genome. We also have boutique libraries covering all the genes known to be involved in apoptosis, cell polarity, kinases, and cell cycle and growth, and we can do discovery-type screens to identify new genes that might impact on those pathways.”

The centre will soon provide rapid sequencing of suspected oncogenes emerging from researchers’ assays, using the latest next-generation sequencing platform, to be housed at Peter Mac.

Simpson says the centre offers Australian researchers a world-class service, comparable to those of centres at Harvard University, Stanford University, the Netherlands Cancer Institute, and the University of Texas Southwestern Medical Centre in Dallas.

The University of Queensland’s Institute of Molecular Bioscience and the Diamantana Institute for Cancer, Immunology and Metabolic Medicine in Brisbane currently operate the only comparable service in Australia. Simpson says the two facilities are collaborating closely to establish standard operating procedures. ---PB---

Complementary technologies

The lentivirus hairpin RNA platform allows researchers to infect primary tumour cells and cell lines – often a difficult thing to do – for long-term studies of cell behaviour in assays such as colony formation and tumorigenesis in agar media.

The siRNA platform provides transient gene knockdown lasting around 72 hours – long enough to observe significant phenotypic changes in cells – with peak knockdown occurring after about 24 hours.

The two technology platforms are complementary. The siRNA transient knockdown platform allows researchers to sift for suspect genes based on transient phenotypic changes in cell lines. They can then use shRNA constructs to knockdown cell lines, and confirm or exclude suspect genes as oncogenes through longer-term studies.

“With breast cancer, for example, we have used siRNA to look for changes in two-dimensional cellular morphology,” Simpson says.

“We then used shRNA to knock down the same gene in cell lines and observe any changes towards breast tumour phenotypes using a three-dimensional morphology assay that replicates the cellular structures that look like of the mammary gland.”

Simpson began collaborating with Dharmacon while working at Harvard in 2004, when the Thermofisher subsidiary was emerging as a leader in siRNA technology.

“Since then, Dharmacon has created RNAi Global, a global research consortium of all institutes that have purchased the whole genome siRNA collection,” she says.

“We hold monthly teleconferences, and hold formal meetings every six months to review progress and discuss new assays and validation techniques developed by members.

“Dharmacon is a very insightful company, because setting up this sort of system involves foregoing a lot of IP opportunities.

“They inform consortium members when they have new prototype products, and we get to beta-test new technologies and feed back information to the company, in return for discounts when they are commercialised.”

Simpson says to screen Dharmacon’s chemically synthesised double-stranded 21-mer siRNA nucleotides on a one-gene-per-well basis requires high throughput robotics.

The centre has acquired the technology, covering the entire 21,000-gene genomes of human and mouse, with ACRF funding.

siRNAs are available in 96- or 384-well plate formats. Each well contains four knockdown sequences, called a SMARTpool, targeting different regions of the gene transcript, to ensure efficient knockdown while minimising off-target effects.

Researchers can assess their screen visually by imaging cells on plates using an automated high throughput microscope, or rely on an automated plate reader to score hits on the basis of the fluorescence from any range of markers.

Simpson says a researcher will typically design an experiment around a series of assays, check that the methodology will yield a statistically rigorous result using genes that positively and negatively influence the phenotype they are following, and then use the centre’s robotic dispenser to deliver the cells and reagents into the wells.

After the transfection step, the robotic system moves the plates directly into an incubator, minimising the risk of spurious effects that can arise in manual handling in different laboratory environments.

The researcher screens the SMARTpool library and analyses the data for statistically significant results. They then repeat the assay with all four RNAi knockdown sequences in individual wells to validate the positive result. ---PB---

Screening for genes

While working at Harvard, Simpson used this high throughput screening and microscopy-based approach to identify 64 genes that regulate cell motility in mammary epithelial cells.

She is now working to determine if these genes play a role in metastatic breast tumour progression. Last year, Simpson published her results in an article entitled ‘Identification of genes that regulate epithelial cell migration using an siRNA screening approach’ in September’s issue of Nature Cell Biology.

“Other researchers have used this technology to identify subsets of genes that regulate susceptibility to HIV and melanoma progression or to identify strong therapeutic targets for specialised drugs,” she says.

She says the automated screening approach represents a fundamental change in mindset from the traditional single gene hypothesis-driven approach.

“You never know what you might shake out – for example, my assay identified a significant number of genes that had not previously been associated with regulation of cell migration.

“The assays can give a variety of results, of which some will obviously not be directly relevant to cancer, but together, you’re building up a bigger picture, bit by bit.

“The automated imaging technology is evolving very rapidly – you can extract multiple components from a single screen. You can use up to half a dozen fluorophores, and capture multiple parameters – it’s up to the researcher’s imagination how they exploit it.”

Simpson says the automated imaging technology generates massive amounts of data, and the centre is buying its own stand-alone server to handle the load.

“When you’ve completed the screens, and done the imaging, the question is how you deal with the analysis – the bioinformatics is very challenging,” she says. “But this technology is an extraordinarily powerful way of searching for genes that strongly influence cancer cell phenotypes, and accessory cancer genes.”