Victorian Centre for Functional Genomics

“Enabling discovery and driving translational medicine with high throughput technologies”

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The Victorian Centre for Functional Genomics (VCFG) at Peter Mac enables biomedical researchers Australia-wide the ability to perform novel discovery-based high throughput screens using multiple platforms. The VCFG facilitates functional interrogation of all genes in the genome, or selected boutique collections using CRISPR/cas9, small interfering RNA (siRNA), micro RNA (miRNA), long non-coding RNA (lncRNA) and short hairpin RNA (shRNA) approaches. The VCFG enables large and small scale drug screens using commercially available compounds or BYO compounds. The VCFG has extensive experience in cellular phenotyping using quantitative high content screening and has developed sophisticated analytical pipelines for data analysis.

The VCFG has established a high throughput 3D pipeline for embedding cell lines, patient- or PDX-derived material and co-culturing with interacting cell types. This work can be performed in both 96 and 384 well format using Matrigel and our robotic automation, or customisable hydrogel 3D using our Rastrum 3D bioprinter instrument from Inventia Life Science. Using our established drug screening pipelines we can transfer this technology to drug discovery in 3D.

The VCFG has an Australia-first, Reverse Phase Protein Array platform, a high throughput platform to rapidly quantify expression of native and phospho-specific protein isoforms in very small sample populations. We have a large antibody collection that can be screened in a customised format or the entire collection at once. We welcome researchers to bring their own antibodies.

The VCFG primarily operates a ‘researcher driven, staff assisted’ model whereby the researcher is embedded in the facility, trained on appropriate equipment and fully supported by the VCFG team from assay concept design, through screening and analysis. Importantly each project is customised to your specific biological question, built on over 10 years experience, we will help drive your project to the best screen outcome possible.

The VCFG is headed by A/Prof Kaylene Simpson and a team of highly experienced research team.

Platforms

3D platform

Using our Janus G3 robot, we can embed cells in Matrigel in 96 well format and using Rastrum cells are embedded in customised hydrogels (bioinks) of varying composition and stiffness in both 96 and 384 well format. This platform enables highly complex co-culture interaction studies, including tumour and immune cells, host-pathogen interactions, angiogenesis and cell invasion. It has great potential for the new era of personalised medicine.  We are currently performing large and small scale compound screening coupled with sophisticated imaging in label-free and fluorescent settings. We have developed a customised analysis pipeline that can measure structure size and viability and importantly, can measure hundreds of unbiased cellular features to cluster structures into groups with like phenotypic signatures. This powerful method can rapidly transform the identification of a large ‘hit list’ into a focused sub-set.

CRISPR/Cas9 screening

The CRISPR/Cas9 system enables gene deletion directly at the genomic level and provides a parallel platform for gene perturbation complementary to RNAi. This approach uses the bacterially derived enzyme Cas9 and a gene-specific single guide RNA molecule (sgRNA) to introduce nicks in precisely defined genomic locations leading to a complete gene knockout.

Pooled viral CRISPR screening

The VCFG houses the latest generation of genome-wide pooled CRISPR libraries to knockout protein coding genes (human, Brunello library and mouse, Brie library), over-express using CRISPRa (Calabrese and Caprano) and inhibit using CRISPRi (Docetto and Dolomiti). To identify genes responsible for the phenotype, we use next-generation sequencing, and together with the Molecular Genomics core we offer a full sequencing pipeline and subsequent data analysis and hit identification. The VCFG also has the TransEDIT dual guide viral library in arrayed format that can be used for single target optimisation or validation, or boutique pooled library screening. Constructs can be cherry picked with short notice and a glycerol stock provided for you to move straight into virus production. We have a number of controls, cas9 protein and nucleofection reagents ready to go in the lab and we facilitate purchasing guides at discounted rates.

Arrayed synthetic CRISPR screening

The synthetic CRISPR platform enables gene knockout in an arrayed format using liquid handling automation in 96 or 384 well format. This platform uses synthetic purified sgRNAs that are transiently delivered into the Cas9-stable cells using lipid transfection reagents, and the phenotype is assessed typically within 72-96h post-transfection. This platform enables cell phenotyping approaches using high content imaging or FACS profiling. Suited to working with both adherent cells through lipid-based transfection or suspension cells through nucleofection.

siRNA, miRNA, long non-coding RNA platform

The siRNA platform is based on Dharmacon's siGENOME SMARTpool reagents arrayed in either 96 or 384 well plate format. siRNAs targeting the protein coding genome, miRNAs (mimic or inhibitor) or long non-coding RNAs are transiently introduced into cells using lipid-based reagents and assayed between three to seven days post-transfection.

Screeners can perform imaging based high throughput, high content cellular analysis, or fluorescence/luminescence based phenotypic quantitation using a high throughput plate reader. Often, researchers multiplex their screens and use both platforms.

Compound Screening

Using high throughput liquid handling automation, the VCFG enables all types of compound screening approaches from cell-based screens to protein complex interaction screens in both 2D and 3D format. From straightforward dose curves with BYO drugs and multiple cell lines to large compound collections there is great creativity in this screening approach and we work with you to develop the assay readouts. We have developed pipelines for generating both 2- and 3-way synergy measurements in 2D and 3D conditions. Compounds are primarily sourced from Compounds Australia Griffith.

shRNA platform

In situations where gene deletion is considered less desirable, the VCFG has a pooled shRNA screening platform using a lentiviral shRNA-mir30 vector system (pGIPZ) to enable stable knockdown in primary, non-dividing and standard cell lines. shRNAs can be screened singularly for validation purposes or screened as pools. The VCFG offers genome-wide and custom screening options and together with the Molecular Genomics core has developed next-generation sequencing pipeline to identify the genes responsible for the biological phenotype of an assay.

Reverse Phase Protein Array platform

Based on the highly sensitive Zeptosens quantitative proteomics platform, researchers extract protein lysates from cell lines or tissue samples using a proprietary buffer system, submit them to the VCFG and select from over 200 native and phospho- antibodies covering major signalling pathways (the collection is continually expanding). Proteins are spotted onto proprietary glass slides, with 66 protein lysates spotted per grid. Each chip can accommodate 6 grids, which translates to 6 antibodies per chip. Researchers can customise their project from the full antibody list or select to screen one of the pre-defined discovery panels (28 and 58 antibodies). The VCFG has a bioinformatics pipeline for quantitative analysis of expression levels and all data is provided.  

For more details contact A/Prof Kaylene Simpson. The VCFG is co-funded by the University of Melbourne, Melbourne Collaborative Research Infrastructure Program, Phenomics Australia, an NCRIS funded consortia and Peter Mac. 

Resources

Available resources

CRISPR

  • Human whole genome arrayed sgRNA library (Horizon Discovery)
  • CRISPR KO Brunello (human) and Brie (mouse) pooled libraries
  • CRISPRa Calabrese (human) and Caprano (mouse) pooled libraries
  • CRISPRi Docetto (human) and Dolomiti (mouse) pooled libraries
  • TransEDIT dual guide whole genome human arrayed viral library
  • Library amplification protocols and reagents
  • In-house NGS and analysis pipelines
  • Arrayed synthetic single crRNA kinome
  • Cas9 and dCas9 virus, Cas9 protein
  • Nucleofection reagents

RNAi

  • Protein coding siRNA (arrayed, whole genome or boutique screens)
  • human and mouse - siGENOME and siON-TARGET-plus libraries
  • Pooled and arrayed shRNA-mir30 whole genome library
  • miRNA over-expression and inhibition (human)
  • Long non-coding RNA siRNA (human)

Compounds

Quantitative Proteomics (RPPA)

  • Over 200 validated antibodies (primarily directed to human target genes raised in rabbit but majority have mouse reactivity)
  • Customise your screen or chose from 2 different pre-defined Discovery target panels
  • Zeptosens lysis buffer
  • InCELL western screening capabilities

Available instrumentation

  • High-content microscopy using fully automated Cellomics CX7 and CX7-LZR, widefield, confocal, live-cell imaging
  • Incucyte SX5 live imaging, metabolism module
  • Cytation5, a sophisticated plate imager and reader
  • Caliper ALH3000 liquid handling robot for all transfection and speciality liquid handling applications
  • JanusG3 liquid handling robot
  • Rastrum 3D bioprinter
  • BioTek 406: three units that aspirate and dispense media, cells, fix and stain and add drugs
  • BioTek multi-flo: aspirate and dispense any plate type
  • Tecan D300E drug dispenser
  • AMAXA 4D nucleofector and 96 well adaptor for high throughput
  • Seahorse metabolic bioanalyser
  • Zeptosens RPPA reader
  • GeSim nanoplotter
  • LiCONICs 220 stacking tower incubator
  • EVOS Fl, bench top fluorescent microscope
  • Barcode printer; all instruments are barcode-read enabled
  • Dedicated and fully backed up server for data and image storage

Access and contact information

Each screen is unique and the VCFG encourages researchers to work in the most biologically appropriate cell line and to think broadly about the scope of their screen. 

The VCFG team works with researchers each step of the way, through assay development, optimisation, transfection and analysis. This partnership begins by working with the laboratory head to discuss the screen and all related logistics, then obtaining reagents, moving into the lab to be trained and alongside us until project completion. A comprehensive user guide and associated instrument guides are provided.

The nature of screening and assay development dictates screen slots cannot be locked in until an assay has passed rigorous statistical criteria. The VCFG team work with researchers to schedule screens appropriately depending on downstream assay needs and there is sufficient instrumentation for operational redundancy to offer a substantial number of screen slots a week. 

All data generated remains the intellectual property of the screener.

Facility staff

A/Prof Kaylene Simpson - Head ORCID LinkedIn

Dr Susanne Ramm - 2iC - compound screening, 3D ORCID LinkedIn

Dr Twishi Gulati - CRISPR screening and RPPA ORCID LinkedIn

Jennii Luu - Lab manager, automation specialist ORCID LinkedIn

Karla Cowley - High content imaging and analysis ORCID LinkedIn

Arthi Macpherson - RPPA operations and analysis ORCID LinkedIn

Robert Vary - Equipment training, screen support ORCID LinkedIn

acknowledgement

The VCFG is funded by:

VCFG Publications and Resources

 can be downloaded here. For VCFG publications list and other resources, please visit our publications and resources page.