Director: Dr. Qiangrong Liang
Staff: Tricia Krueger (RA) and Yuan Huang (RA)
Mission Statement:
The goal of Core D is to provide high quality adenoviruses to laboratories in a timely fashion .
Current Services:
1. Construction of recombinant adenoviruses
2. Amplification, purification and tittering of adenoviruses
Future Service: The Core is developing techniques
1. For the construction of adenovirus encoding short hairpin RNA (shRNA).
2. For the amplification of Adeno-Associated Viruses (AAV).
Check back later for the availability of these services.
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.
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Guidelines for Submitting Constructs for the Molecular Biology Core (Core D)
To Create Recombinant Adenoviruses
1. In order for the Core to create viruses, a pShuttle vector harboring the gene of interest is expected to be submitted
a. In general, creation of a recombinant adenovirus requires 3 major steps. First, construction of pShuttle vectors containing the gene of interest for homologous recombination in bacteria. Secondly, generation of recombinant adeoviral plasmids by homologous recombination in E.coli.(BJ5183). And finally, production of adenoviruses in HEK293 cells.
b. A gene of interest is inserted into pShuttle CMV vectors or pShuttle vectors at the multiple cloning sites (MCS, see the map of vectors for detail) by following conventional molecular cloning procedures. Please submit your plasmid at a known DNA concentration from 0.5 to 1 ug/ul.
c. Molecular Biology Core provides the pShuttle CMV or pShuttle vectors as well as the foreword and reverse primers used for pShuttle vectors sequencing and PCR.
d. Make sure that your DNA insert does not contain either Pac1 or Pme1 restriction site because these two sites exist in pShuttle vectors and are needed for viral construction. If your insert DNA does contain these sites, it will be necessary to perform site-directed mutagenesis before proceeding to the cloning steps.
2. Evidence of the quality of the plasmid submitted
a. To minimize invalid effort in the subsequent steps of viral creation, the Core expects you to submit the following evidence to show that your pShuttle contains a valid gene of interest:
i. PCR and/or restriction enzyme digestion results that prove that the gene of interest is correctly inserted into the MCS of the pShuttle.
ii. It is highly recommended to verify your insert in the pShuttle by sequencing at least the junction between vector and insert.
iii. A western blot must be submitted along with your pShuttle plasmid to show that the pShuttle harboring your gene of interest expresses the protein expected by transfecting a cell line (e.g. 293 cells) with your pShuttle plasmid. Molecular weight markers and negative controls must be included on the same western.
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