Viral Facility

The facility is setup to cater for the production of lentiviruses and adeno-associated viruses.

Introduction

The Viral Facility is a shared facility which provides the space, training, equipment and other resources to help in the production of replication-deficient viral vectors as well as in-vitro and in-vivo viral experiments.

The facility is built as a PC2 containment area and complies with Australian standards AS2982, AS2243 and AS2647. The facility is located in the Physiology department (North Wing of the Medical School Building) on the 5th level (Rooms N511, N512, N512A and N513).

There are four main areas within the facility:

  1. Anteroom (Rm N512A) is the main entry to the facility with an air-lock system containing a "gowning up" area prior to entering the labs.
  2. Cell Preparation Laboratory (Rm N513) which is used for the propagating cell lines and/or establishing primary cell cultures only.
  3. Viral Preparation Laboratory (Rm N512) which is used for the production, amplification, concentration and purification of viral vector particles.
  4. Viral Experimental Laboratory (Rm N511) which is used for in-vitro and in-vivo viral experiments.

Contacts

Please contact Prof Andrew Allen or Angela Connelly, if you have any issues or questions regarding the Viral Facility.

Prof Andrew Allen
Ph: 61 3 8344 5838
Email: amallen@unimelb.edu.au

Angela Connelly
Ph: 61 3 8344 5580
Email: aac@unimelb.edu.au

Viral Vector Overview

Replication-deficient viral vectors have been successfully used as tools for gene delivery by many laboratories around the world. Some of the advantages of using viral vectors over conventional gene delivery/pharmacological methods are:

  1. Broad or cell-specific targeting both in-vitro and in-vivo
  2. Infect dividing and non-dividing cells.
  3. Stable gene expression that can last for weeks to months (alternative to transgenic models)
  4. Use in many animal models (from rodents to primates).
  5. Diverse range of applications (from basic research to gene therapy).

Viral Vector

Max. insert size

Duration of
Expression

Pros

Cons

Adenovirus

7.5kb

Transient

High titres, safe

Immunogenic, transient

transgene expression

Lentivirus

8kb

Stable

Non-immunogenic,

long-term expression

Low titres, random

integration

(mutagenic)

Adeno-

associated virus

4.5kb

Stable

Specific integration (safe),
non-immunogenic, long-term
expression

Small packaging capacity

Costs

All users will be charged based on the number of virus preps. Cost/viral prep* is as follows:
   Lentivirus = $1280
AAV = $ 990
Additional charges for assistance based on the level of assistance required:
Full = $1075
Partial = $215

*The cost has been calculated to include equipment running servicing and running costs for the facility, including ultracentrifuge, biosafety cabinets, microscopes, Liquid nitrogen, C02, incubators, autoclave, etc. Also included is the average cost of consumables and reagents used for making a specific virus (large scale) according to protocols provided. Rates may also vary depending on changes to the prices of the items, or follow a different protocol that uses more or less of the listed items.

Getting Started

Prior to registering as a user, you must have:

  • The appropriate OGTR licence to work with viral vectors within the facility.  If applicable, you must also have AEC approval for any in-vivo viral experiments performed in the facility and AQIS permit for viral or other GMO imports to be used in the facility.
  • Completed the University of Melbourne's "Biohazard Lab Practice" training or an equivalent training from your institute. You can complete this training online on the University's Gene Technology and Biosafety
  • In order to register as a user and access the facility, you must:

  • Read and familiarise yourself with the viral facility's standard operating procedures and risks involved.
  • Undergo a formal induction by the viral facility manager.
  • Be trained to use the major equipment – Class II Biosafety cabinet, autoclave, ultracentrifuge and fluorescent inverted microscope by an authorised trainer.
  • Be trained to perform viral production and/or viral experimental protocols by an authorised trainer.
  • Bookings

    To make bookings go to Google Calendar.

    List of Consumables/Reagents and equipment available

    Adenovirus

    Lentivirus

    AAV

    Ad.CMV.GFP (AdGo) –

    ubiquitous cytomegalovirus promoter driving GFP

    Ad.GFAP(B)3.nLacZ –

    glial-specific enhanced GFAP promoter driving nuclear LacZ

    Ad.NSE.LacZ –

    neuron-specific enolase promoter driving LacZ

    Ad.tubulinα1.EGFP –

    neuron-specific tubulin-α1 promoter driving EGFP

    Ad.PRSx8.LacZ –

    catecholamine neuron-specific synthetic dopamine β hydroxylase promoter driving LacZ

    Lv.CMV.EGFP –

    ubiquitous cytomegalovirus promoter driving EGFP

    Lv.Ubiquitin.EGFP –

    ubiquitin promoter driving EGFP

    Lv.CaMKII.EGFP –

    pan-neuronal calcium/calmodulin-dependent kinase II promoter driving EGFP

    Lv.SYN1.EGFP –

    pan-neuronal human synapsin 1 promoter driving EGFP

    Lv.CBA.tdTomato –

    pan-neuronal chicken β-actin promoter withcytomegalovirus (CMV) immediate-early enhancer driving tandom dimer tomato (red fluorescent) protein

    Lv.PRSx8.EGFP –

    catecholamine neuron-specific synthetic dopamine β hydroxylase promoter driving EGFP

    Lv.TPH.EGFP –

    serotinergic neuron-specific promoter driving EGFP

    All are serotype 1/2 and contain chicken β-actin promoter with cytomegalovirus (CMV) immediate-early enhancer

    AAV-DCA-EGFP

    AAV-DCA-tdTomato

    AAV-DCA-mOrange

    AAV-DCA-mEGFP

    (membrane bound EGFP)

    AAV-DCA-mtdTomato

    (membrane bound tdTomato)

    AAV-DCA-ERtdTomato

    (endoplasmic reticulum-targeted tdTomato)

    AAV- CBA-ChR2(H134R)mCherry

    (pan-neuronal chicken β-actin promoter withcytomegalovirus (CMV) immediate-early enhancer driving Channel rhodopsin- mCherry)

    PLASMIDS

    Gateway Vector Conversion System (Invitrogen; cat no: 11826-029)
    Multisite Gateway Pro2.0 (Invitrogen; cat no: 12537102)

    Adenovirus Lentivirus  AAV
    Shuttle plasmids:
    pAdTrack (Stratagene)
    pAd-PRSx8-LacZ
    pAd-NSE-LacZ
        
    Packaging plasmid:
    pAdEasy-1
    (Stratagene)
        
    Shuttle plasmid: 
    pXCX-SW-Linker
    Packaging plasmid:
    pBHG10

    Transducing plasmids:
    pTYF-EF1α-linker
    (with elongation factor 1α promoter) 
    pTYF-PRSx8-linker
    pTYF-PRSx8-EGFP
    pTYF-CMV-EGFP
    pTYF-PRSx8 ChR2(E123T-H134R)-YFP WPRE
    pTYF-PRSx8 AstR-IRES GFP
    pTYF-CamKII-hChR2(E123T-H134R)-YFP
    pTYF-PRSx8 Cre
    pTYF-CBA- CreTdtomato WPRE
    pTYF "Gateway Compatible"

    Envelope plasmids:
    pVSVg (vesicular stomatitis virus G)
    pGMok (mokola lyssaviruses virus G) 
    Packaging plasmid:
    pNHP
    Enhancer plasmid:
    pCEP4-tat

    Shuttle plasmids:
    pAM-DCA-tdTomato
    pAM-DCA-mEGFP 
    Packaging plasmids (AAV1/2):
    Helper plasmid pDP1  
    Helper plasmid pDP2

    Packaging plasmids (AAV2):
    pHelper (Stratagene)
    pAAV-RC (Stratagene)

    Restriction maps and sequences provided on request

    Cell lines:
    HEK293FT (Invitrogen; cat no: R700-07)
    AAV293 (Stratagene; cat no: 240073)
    HEK293A (Invitrogen, catno: R70507)