MOLECULAR BIOLOGY CORE
Director: Dr. Qiangrong Liang
Staff: Tricia Krueger (RA)
Mission Statement: The goal of Core D is to provide high quality adenoviruses to laboratories in a timely fashion.
Current Services:
- Generation of recombinant adenoviruses
- Amplification and purification of adenoviruses
Future Service: The Core is developing techniques for the construction of adenovirus encoding short hairpin RNA (shRNA). Check back the availability of this service later.
Order for Core Services
Before placing an order for Core D services, one should talk with Core D staff to discuss your specific needs and to get a pShuttle vector for cloning your gene of interest. Please follow the Guidelines for the Sub-cloning and confirm the gene expression before submitting the construct to the Core.
Progress Tracking:
One can check the progress of the viruses under construction in the order and progress report form, which is located in the X-drive/Projects/Liang lab/Virus Core folder. Generally, it takes 4-5 weeks to produce a viral stock, which can be delivered to the client if high titter amplification is not required. Amplification and Purification will take another 1-2 weeks.
Overview of Adenoviral Construction
Adenovirus is a powerful tool for gene delivery and expression. Adenoviruses are capable of infecting a broad range of cell types and infection is not dependent on active host cell division. An AdEasy Adenoviral Vector System (Stratagene) is used in the Core to make adenoviruses.
The first step is to clone a gene of interest into a shuttle vector such as pShuttle-CMV. This vector contains a multiple cloning site between the CMV promoter and the SV40 polydenylation signal and is suitable for insertion of a cDNA as big as 7 kb.
Once the gene of interest is inserted into the vector, it is linearized by restriction digestion using Pme1 enzyme. It is then transformed by electroporation into BJ5183 cells that contain the pAdEasy-1 plasmid encoding the Adenovirus-5 genome. The homologous recombination between pShutte-CMV and pAdEasy-1 in BJ5183 cells allows the insertion of the gene of interest into the adenoviral genome.
The successful recombinant plasmid is confirmed by restriction digestion with Pac1. It is then be electroporated into DH10B cells for amplification. The purified DNA is transfected into Ad293 or HEK293 cells for generating a crude viral stock.
The crude viral stock can be further amplified and purified by CsCl banding or using the Adenopure Adenovirus Purification Kit (Puresyn).
