Viral Core Facility (VCF)
The Viral Core Facility (VCF) offers Charité scientists and external academic scientists ("non-profit Organizations") services for the generation of viral particles.
The Viral Core Facility services covers three areas. The first area includes a DNA service with the production of new viral shuttle vectors and purification of DNA for cell transfection. To this end, there is a consultation on the individual vector systems that can be provided by the VCF. If desired, users can clone their shuttle vectors in their own S1 facility. The second area involves the production of viral particles for in-vitro or in-vivo transductions. In the third area, different virus quantification methods are offered. The following table summarizes the various services.
The researchers choice for the most suitable viral vector depends largely on the specific question the researcher likes to address and in turn on the individual properties of the virus. The VCF offers 4 types of viruses, which differ in their packaging capacity, tissue tropism and their ability to infect dividing and non-dividing cells. The following table summarizes the basic properties of the available viral vectors.
Viral vectors offered by the VCF
You can choose from these types of vectors for your specific needs or you provide us with already made viral vector DNA for production. If you would like to use any of the listed vectors you need to possess specific MTAs (like Addgene MTAs) or other approvals for using specific sequences. Please contact us for specific inquiries regarding required MTAs and choice of vectors which could suite best for your research.
For the generation of adenoviruses we are currently using kits from
Clontech. The viral vector is generated by enzyme mediated insertion
directly from a purified PCR product of the transgene into the the E1
and E3 deleted Ad5 genome. Transgene expression is driven by the strong
Viral production is performed by transfection of the linearized Ad-vector into HEK293 cells expressing E1 and E3 genes of the Ad5 genome and several rounds of amplification. Alternatively, existing adenoviruses can simply be used to start the amplification. Purification of properly assembled adenoviral particles is done by caesium chloride density gradient followed by dialysis.
For generating adeno-associated viruses you can choose from 3 shuttle vector types. All contain 5’ and 3’ AAV ITRs and a WPRE. In the basic vector you can express the transgene by using a variety of different promoters. In the moment we are using CamKII, hSynapsinI, CMV early enhancer/chicken β actin (CAG), phosphoglycerate kinase (PGK), EF-1 alpha, CMV and a liver specific promoter (LP1).
In the second set of shuttle vectors we apply LoxP sequences and an invert-oriented transgene to allow specific expression in Cre-transgenic animals. Here the same promoter settings are available as in the first vector type.
In the third set of AAV shuttle vectors we apply the hSynapsin1 promoter together with reporter proteins coupled via a 2A sequence to a MCS into which the transgene can be cloned. This configuration allows a bicistronic expression of reporter gene and transgene through one promoter.
We utilize helper plasmids providing adenoviral genes necessary to drive AAV replication (E2a, E4, VARNA). In order to achieve tissue specific expression we are applying specific AAV serotype helper plasmids which envelope the AAV capsid for the Serotypes 1,2,3,4,5,6,8,9 and rh10.
The underlying purification method depends on the serotype of the generated AAV. Heparin-columns are used for AAV2/2 particle purification. Iodixanol gradiends are used for the others. If necessary HPLC purification methods are used for AAV2/8 (in cooperation with AG Weger, Institute of Virology, Charité).
For external users, please contact us for specific MTAs for the plasmids provided by Penn Vector Core (for Serotype 9, rh10).
As retroviral vectors we are currently using a MSCV (murine stem cell virus) based retroviral expression system, in which a 5’LTR from the murine stem cell PCMV virus drives high-level, constitutive expression of the transgene in stem cells and other mammalian cell lines.
For generating puromycin resistant cell lines you can choose a second
type of vector, which contains a PGK promoter driven puro expression
Retroviral production is performed by transfection of the retroviral vector into the packaging cell line BOSC23; a HEK293T cell line into which retroviral packaging functions of Moloney Murine Leukemia Virus (MoMLV) were stably introduced. Using this cell line we can generate high titers of ecotropic retroviral particles, which stably integrate into proliferating mouse and rat cells (S1 Level).
All vectors are based on the lentiviral shuttlevector FUGW from Lois et al. 2002. In this basic vector we exchanged the promoter and included a multiple cloning site (MSC). For now we are using CMV, hSynapsinI or hUbiquitin C Promoters for driving the expression of the transgenes.
In a second type of vectors we employed different reporter genes together with a self-cleaving 2A peptide from porcine teschovirus-1 (P2A) followed by the MCS. This allows us the bicistronic expression of reportergene and transgene through one promoter.
The third set of viral vectors utilizes the combination of two different Lox sites. The introduction of a reporter gene plus a reverse oriented reporter gene in conjunction with P2A and MSC make these vectors suitable for cell-type specific expression of the transgene in Cre transgenic animals.
For reducing endogenous mRNAs levels we use the following viral vector which contains the human RNA polymerase III promoter U6 for driving the transciption of specific shRNAs. A second promoter (hUbiquitin C or hSynapsin1) drives the expression of different reporter genes.
Service costs and ordering
In the following tables the individual services of the VCF are shown. As the core facility is a non-profit service supplier of the Charité – Universitätsmedizin Berlin, the service costs are compensations for expensive like cost for used material and personnel.
The services are divided into vector service (1), production (2) and quantification (3) of the different types of viral particles. If users intend to clone the shuttle vector, the scientific management offers an advisory service. The costs are net prices plus all applicable taxes such as VAT.
We had to adjust the costs for AAVs due to its time consuming workflow.
We are pleased to provide research material and reagents from the research labs of the VCF.
Luciferase reporter plasmids
Based on the plasmid pGL3 we offer highly purified plasmid preps for luciferase reporter assays.
For interested neuroscientists, who want to work with an in-vitro autaptic culture system, we are offering microdot plates in 6 well and 12 well formats. Each well contains a cover glass on which 300 µm diameter dots are stamped which allow astrocytes and neurons to grow.
(image was provided by Dr. Rost, upper left corner - stamp for microdots, lower left corner – microisland culture (scale 200 µm), right – a neuron growing on a glia island (scale 50 µm))
For ordering viral particles and using the services provided by the VCF, you have to accept our user rules.
Please download and fill out the specific order form using Adobe Reader.
Order form for adenoviral particle services.
Order form for adeno-associated viral particle services.
Order form for retroviral particle services.
Order form for lentiviral particle services.
For ordering side product of the VCF please use the following order form:
Order form for luciferase reporter plasmid preperations.
Order form for microisland cell culture plates.
Once you send the order form to us, we will contact you as soon as possible.
Contact of the VCF
The VCF is located within the Charité Campus Mitte, Charite-Cross-Over, Virchowweg 6, Level 3 (Rosenmund/Schmitz lab area). Adenovirus and retrovirus production is performed in the Center for Cardiovascular Research (CCR), Hessische str. 3-4, Schupp laboratory. You can contact us by using the following email adress: firstname.lastname@example.org
Dr. Thorsten Trimbuch
Head of VCF and
Bioengineer Anke Schönherr
Dipl.Ing. Matthias Münzner
The research technicians, who help to process your specific needs, are:
The User Advisory Council is constituted by:
|Prof. Christian Rosenmund||Prof. Dietmar Schmitz||Prof. Michael Schupp||Prof. Britta Eickholt|
|The Viral Core Facility is listed at RIsources, the Research Infrastructure Portal of the German Research Foundation DFG.|