The Molecular Biology and Virus Production (MB/VP) Module generate and validate DNA constructs primarily for use in mammalian cultures/systems in vision research. This module also purifies and concentrates 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 provides 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 supports a range of enabling recombinant DNA technologies and provides the assistance and expertise required to test unique hypotheses posited by vision scientists at UTSW and neighboring institutions.