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 is a national platform that 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 in both 2D and 3D. The VCFG has extensive experience in cellular phenotyping using quantitative high content screening and has developed sophisticated analytical pipelines for data analysis.

The VCFG provides a collaborative and innovative partnership. We primarily operate a ‘researcher driven, staff assisted’ model where we work with you each step of the way, through assay development, optimisation, transfection and analysis. This partnership begins with a discussion with A/Prof Kaylene Simpson followed by embedding into the laboratory, training on instruments and performing experiments alongside us until project completion. Comprehensive user guides and associated instrument guides are provided. All data generated remains the intellectual property of the researcher. Importantly, each project is customised to your specific biological question, 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. A/Prof Kaylene Simpson can be contacted on [email protected].

NEW TO THE VCFG!

Equipment

Through partnership with Trend Bio, we recently installed the IsoLight instrument from IsoPlexis. The instrument is capable of single cell and bulk proteomics, with an emphasis on immuno-oncology applications. Researchers can map the adaptive or innate immune landscape of human and mouse T cells, CART cells and NK cells by simultaneously measuring up to 32 different cytokines secreted by each single cell. It can perform fully automated multiplex ELISAs of up to 30 cytokines per sample in over 60 samples in one run. Researchers can identify tumour signalling pathways associated with drug resistance or metastasis by investigating intracellular phosphoproteins expressed in single cells.

VCFG PRoject Information and Management Enterprise (PRIME) 

Every project is securely managed through PRIME, our project management portal using the Atlassian tool Confluence. Analysis is provided in a fully interactive HTML format with all data downloadable, all code easily accessible and representative images available. All funding and outputs (abstracts, presentations, publications) are collated for future reporting. This portal future-proofs our capacity to revisit your dataset for publication, for re-analysis or for large-scale sharing.

Grants

We proudly support your grant applications through developing a customised approach to your specific project and budget. Happy to discuss your ideas! Email A/Prof Kaylene Simpson if interested.

Platforms

3D Culture, Characterisation and Screening

The VCFG has established a high throughput 3D pipeline for embedding cells in Matrigel using our robotic automation or customisable hydrogel 3D using our Rastrum 3D bioprinter instrument from Inventia Life Science. This platform enables highly complex co-culture interaction studies, including tumour and immune cells, host-pathogen interactions, angiogenesis and cell invasion. We routinely perform large- and small-scale compound screening coupled with sophisticated daily imaging in label-free conditions and/or high multiplex fluorescent marker staining as an end point. We have developed a customised analysis pipeline for cell lines, PDX-derived patient cells or directly derived patient materials, allowing us to begin to make dissect cellular responses and make therapeutic predictions (Choo et al 2021). For a broader overview and some FAQs see our short explainer.

Resources available:

Matrigel (sourced in large volumes to provide consistent lot numbers).

CRISPR Platform

CRISPR screens enable researchers to determine resistance in their cellular model, facilitate biomarker discovery in patient samples or determine genes responsible for synthetic lethality. VCFG supports both synthetic and pooled CRISPR screening strategies. More details on this platform can be found below and on our short FAQ explainer.

Synthetic CRISPR Platform

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 Cas9-stable cells using lipid transfection reagents or nucleofected with purified Cas9 protein. The phenotype is assessed typically within 72-96 hours. This platform enables cell phenotyping approaches using high content imaging, fluorescence/luminescence plate reader end points or FACS profiling.

Resources available:

  • Human whole genome arrayed sgRNA library (Horizon Discovery)
  • Arrayed synthetic single crRNA kinome
  • Cas9 virus or protein
  • Nucleofection reagents
  • Purchasing sgRNAs at discounted rates

Viral CRISPR Platform

The VCFG houses both pooled and arrayed viral CRISPR libraries. Pooled CRISPR library are analysed using next generation sequencing. We take you through the screening process step-by-step for both whole genome pooled library or custom pooled sublibrary screens, from planning to execution through to analysis. The TransEDIT dual guide viral library in arrayed format can be used for single target validation, or boutique pooled library screening.

Resources available:

  • 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
  • Custom pooled CRISPR sublibrary generation and reagents
  • Library amplification protocols and reagents
  • In-house NGS (in collaboration with Molecular Genomics Core) and analysis pipelines
  • Cas9 and dCas9 virus
  • TransEDIT dual guide whole genome human arrayed viral library

RNAi Platform

siRNA, miRNA, long non-coding RNA Platform

The siRNA platform is based on Horizon Discovery'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 72-144 hours post-transfection. This platform offers similar readout options as screening arrayed CRISPR.

Resources available:

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

shRNA Platform

The VCFG has a pooled shRNA screening platform to enable stable knockdown in primary, non-dividing and standard cell lines. shRNAs can be screened singularly for validation purposes or screened as genome-wide or custom pools. The VCFG together with the Molecular Genomics Core has developed the next-generation sequencing pipeline to identify the genes responsible for the biological phenotype of an assay.

Resources available:

  • Pooled and arrayed human shRNA-mir30 whole genome library

Compound Screening

Using high throughput liquid handling automation, the VCFG enables all types of compound screening approaches from cell-based to cell-free systems. We can deliver compounds in both 2D and 3D format and can accommodate straightforward dose curves with BYO drugs in multiple cell lines, 2- and 3-way drug synergy and large-scale collections. There is great creativity in this screening approach and we work with you to develop the most appropriate experimental design and assay readouts. For a broader overview and some FAQs see our short explainer.

Resources available:

High Content Imaging

Arrayed screening in the VCFG has always been underpinned by our strengths in quantitative cellular phenotyping, where we develop and apply sophisticated custom analysis tools to cells stained with multiple phenotypic markers. In recent years, this approach has become relatively routine in the VCFG for screens performed in 2D. During the pandemic we invested significant R&D to develop methods to quantify structures grown in matrices in 3D, where we focused not only on expanding our bright-field imaging platform but also to develop methods to fix and stain with multiple fluorescently labelled cell markers and quantify complex phenotypes.  We now can offer a suite of cellular machinery stains and associated analytical tools for use in both 2D and 3D. See our high contect imaging explainer for more details.

Instrumentation

  • AMAXA 4D nucleofector and 96 well adaptor for high throughput
  • Barcode printer; all instruments are barcode-read enabled
  • 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
  • Caliper ALH3000 liquid handling robot for all transfection and speciality liquid handling applications
  • Cytation 5 plus BioSpa, a sophisticated plate imager and reader with live cell imaging
  • Cytation C10, a sophisticated imager with spinning disk confocal and plate reader
  • EVOS Fl, bench top fluorescent microscope
  • High-content microscopy using fully automated Cellomics CX7-LED and CX7-LZR, widefield, confocal, live-cell imaging
  • Incucyte SX5 live imaging, metabolism module
  • IsoLight from Isoplexis
  • Janus G3 liquid handling robot
  • LiCONICs 220 stacking tower incubator
  • Rastrum 3D bioprinter
  • Seahorse metabolic bioanalyser
  • Tecan D300E drug dispenser
  • Dedicated and fully backed up server for data and image storage

Facility staff

A/Prof Kaylene Simpson - Head ORCID LinkedIn

Dr Susanne Ramm - 2iC - 3D organoid characterisation, screening, analysis ORCID LinkedIn

Dr Mark Li- 3D screen support and fee for service, analysis ORCID LinkedIn

Jennii Luu - Lab manager, automation specialist, 2D and 3D platforms ORCID LinkedIn

Karla Cowley - High content imaging and analysis, 2D platform ORCID LinkedIn

Dr Henry Beetham - CRISPR screening, MRFF stem cell project, 2D screens and analysis ORCID LinkedIn

Dr Twishi Gulati - Phenomics Australia In Vitro National Service Coordinator, iLAB management, business development ORCID LinkedIn

Arthi Macpherson - Equipment training, new instrumentation, PRIME management ​​​​ORCID LinkedIn

Robert Vary - Equipment training, screen support, fee for service projects ORCID LinkedIn

acknowledgementS

VCFG has infrastructure funding and support from

  
 
 

Researchers accessing the VCFG have funding from

 

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Since 2008, the VCFG has been a member of 4 successful ACRF infrastructure program applications

VCFG Publications and Resources

For VCFG publications list and resources, please visit our publications and resources page.