The Molecular Biology and Virus Production (MB/VP) Module will generate and validate DNA constructs primarily for use in mammalian cultures/systems in vision research. This module will also purify and concentrate both lentivirus and adenovirus for subsequent use in immortalized and primary cultures (maintained by individual investigators and the Cell Culture & Cell Phenotyping Module). The MB/VP Module will provide the following services: 1) De novo generation of constructs to be used for vision research 2) modification (mutagenesis, insertion/deletion) of plasmid DNA; 3) maintenance and propagation of DNA glycerol stocks; 4) production and purification of lentivirus and adenovirus; 5) validation of established DNA constructs and lentivirus/adenovirus by immunocytochemistry, qPCR and western blotting.
The specific services provided within the MB/VP Module are:
- Site-directed mutagenesis for generating point mutations, insertions, or deletion variants
- Custom Gibson assembly for development of new DNA fusion constructs driven by desired promoters of interest
- ShRNA and CRISPR/SpCas9 plasmid generation for knockdown/out experiments
- Full sequencing of generated constructs, extensive documentation, and maintenance of DNA stocks for future use
- Small to large scale endotoxin free plasmid purification for downstream applications
- Thorough confirmation of produced constructs at the transcript, protein and subcellular level
- Generation of high-purity lentivirus and adenovirus for use in vitro or in vivo
Overall, the MB/VP Module will support a range of enabling recombinant DNA technologies and provide the assistance and expertise required to test unique hypotheses posited by vision scientists at UTSW and neighboring institutions.