“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 with 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 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.
The VCFG has installed the first 3D bioprinting instrument from an Australian company, Inventia Life Science. The Rastrum can embed cells from cell lines or patient tissues and enable complex interaction studies using a matrix scaffold that can support 3D growth. Using our established drug screening pipelines we can transfer this technology to drug discovery in 3D.
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. Fee for service contract-based screening can also be considered. The VCFG is headed by A/Prof Kaylene Simpson.
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 and mouse Brie library), over-express using CRISPRa (human Calabrese and mouse Caprano library) and inhibit using CRISPRi (human Docetto and mouse Dolomiti library). 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.
Arrayed CRISPR screening
The VCFG supports both viral and synthetic arrayed CRISPR screening. The VCFG 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.
The synthetic CRISPR platform enables gene knockout in an arrayed format using similar methodology to siRNA screening 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.
The VCFG houses the NucleofectorTM Technology that enables highly efficient transfection of primary cells, stem cells, neurons, and cell lines that have traditionally been difficult to transfect via electroporation and other non-viral transfection methods. It is a commonly used method to deliver Cas9/sgRNAs ribonucleoproteins (RNPs) to your target cells.
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.
Using high throughput liquid handling automation, the VCFG enables all types of compound screening approaches from cell-based screens to protein complex interaction screens. 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. Compounds are primarily sourced from Compounds Australia Griffith.
Using the Rastrum technology, we can embed cells in customised hydrogels (bioinks) in high throughput applications by varying composition and stiffness. This enables exploration of cell lines, patient tissues and iPSCs to derive a picture of growth kinetics, response to targeted gene engineering and drug discovery. 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.
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.
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 RPPA platform is co-funded by the University of Melbourne, Melbourne Collaborative Research Infrastructure Program together with partners Bio21, University of Melbourne Department of Anatomy and Neuroscience, Murdoch Children’s Research Institute and Peter Mac.
- CRISPR KO Brunello (human) and Brie (mouse) pooled libraries
- CRISPRa Calabrese (human) and Caprano (mouse)
- CRISPRi Docetto (human) and Dolomiti (mouse)
- 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
- 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)
- Accessed from Compounds Australia Griffith
- BYO compounds welcome
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
- Capacity for western blotting validation using the LiCOR Odyssey and accessing the antibody collection
- InCELL western screening capabilities
- High content microscopy using fully automated Cellomics CX7 and CX7-LZR, Arrayscan VTi, widefield, confocal, live-cell imaging
- Rastrum 3D bioprinter
- Caliper ALH3000 liquid handling robot for all transfection and speciality liquid handling applications
- JanusG3 liquid handling robot
- 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
- Cytation5, a sophisticated plate imager and reader
- Tecan D300E drug dispenser
- Seahorse metabolic bioanalyser
- LiCOR Odyssey CLx infra-red detection system including InCell western module
- Zeptosens RPPA reader
- GeSim nanoplotter
- AMAXA 4D nucleofector
- 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.
The VCFG is funded by:
- the Australian Cancer Research Foundation (ACRF)
- the University of Melbourne, Melbourne Collaborative Research Infrastructure Program
- the Australian Phenomics Network (APN) through the National Collaborative Research Infrastructure Scheme
- the Victorian Department of Economic Development, Jobs, Transport and Resources
- the NHMRC
- Cancer Council Victoria
- the Jack Brockhoff Foundation
- the Peter MacCallum Cancer Foundation
- Tour de cure