Personnel:
Paul R. Kinchington, Director (contact Director: Kinch@pitt.edu)
Miguel Betegon, Co-Director
Leah Byrne, Co Director
Full time Technical Specialist and Staff Scientist; Shenghuo Tian, PhD.
What we do and can do for you:
The VPMP module is designed to help faculty in the preparation and purification of Viruses for use in research: and for the manipulation of protein expression in cells, animals and models of ocular disease. The module is a redesign of the former Molecular Biology module that had been part of the P30 Core grant since its inception and first funding by the National Eye Institute in 1988. The New module followed from a survey of participating faculty for their current needs, and many indicated a strong interest in focusing this module on the development and production of viruses or virus particles for gene delivery; for the modulation of protein expression by silencing (virus vector driven siRNA and shRNA techniques, or gene knockout by editing methods, including CRSPR-cas9): or by protein overexpression and gene delivery. Virus vectors familiar to the module Directors and available for use include four plasmid based lentivirus construction, development of herpesviruses, expression in replication competent and defective adenoviruses, and the generation and purification of adeno-associated virus (AAV) of multiple serotypes. The module also has resources needed for virus and protein purification. The virus particles could be developed for a number of applications, including the development of stable cell lines, CRISPR screens, or for the purpose of gene therapy vectors in animal studies. Most labs in the Department are using viruses as tools to manipulate gene expression in cell cultures, organoids, and animals. The VPMP module will accelerate these studies by providing routine services, the centrifugal Equipment and Rotors needed and the use of column based methods of purification. Some faculty have expressed an interest in using AAV vectors for setting up optogenetic studies, and do not have the molecular expertise to do so. The VPMP module fulfills this need by providing expertise in virus production and purification for animal studies.
Key Core Module Equipment:
-Bacterial Shakers and several incubators for Cloning, DNA manipulation and preparation;
-everything for DNA and RNA manipulation and analyses including multiple PCR machines (7), Quantitative PCR analyses by Real Time PCR and Digital Droplet DD PCR) platforms
- NanoDrop and Qbit spectrophotometry devices, Horizontal gel electrophoresis platforms of all sizes, 2 separate Imaging and documentation Systems for DNA based on UV, Sybr Green, and other multiple colors and outputs
-Libraries of Plasmids for different vectors and the expression of various capsid serotypes, lentivirus packaging systems and lentivirus expression Systems
Beckmann Ultracentrifuge with large and small Scale Swing-out rotors, near vertical angled Rotors
Beckmann Benchtop Micro-Ultracentrifuge with titanium Rotors.
- High speed Ultracentrifuge, benchtop high speed ultracentrifuge, 2 mid speed Centrifuges, Temperature cooled sonicators
-All materials and equipment for protein electrophoresis, immunoblot quantification and analyses at multiple wavelengths simultaneously at small, mid and large scale formats.
-4 x Incubators at 37 for tissue culture, with the capacity for suspension cultures of eukaryotic cells under 37oC and 5%CO2
-BSL2 Hood systems with the capacity to be upgraded to BSL2+ vector development conditions.