RNAi Adenovirus Packaging Service

BOC Sciences can construct adenoviral gene overexpression vectors and RNA interference vectors, offering a wide range of options for your experimental design.

Introduction to Adenovirus

Adenovirus (Ad) is a spherical, envelope-free virus with a diameter of approximately 70-90 nm. The capsid is a 20-hedron, stereometric structure consisting of 252 capsid particles, of which 240 are Hexon and 12 are Penton. The fibrous spikes located on the surface of the capsid are based on the penton protein, and 12 fibers protrude from the surface of the capsid. It can bind to receptors on the cell surface and play a very important role in the process of virus infection of cells. The genome of Ad is a linear, non-segmented double-stranded DNA (dsDNA), approximately 25-45 kb in size, consisting mainly of E1a, E1b, E2a, E2b, E3 and E4 (encoding viral regulatory proteins) in the early stages of viral DNA replication and L1 to L5 (encoding viral structural proteins) in the late stages of DNA replication. Ad replicates independently of host cell division and has a wide range of hosts, infecting both dividing and non-dividing cells. It is epitheliophilic and can be endocytosed into cells by binding to specific receptors on the cell surface through the capsid region of its fiber, and then transferred from the endosome to the cytoplasm and nucleus to initiate viral replication and assembly through the cell's transcriptional and translational machinery.

There are 52 known adenoviruses, named Ad1 to Ad52, of which Ad5 is one type of adenoviral vector (AdV) commonly used to transform replication defects, and its genome is a linear 36kb dsDNA molecular. Ad5 is missing the adenovirus early expressed gene sequences E1 and E3, of which E1 is required for adenovirus replication. Thus, E1 deletion prevents Ad from completing its own replication and relies on the trans-complementation provided by the packaging cells for replicative amplification. E3 encodes a cell surface glycoprotein that counteracts the host's antiviral defense system and, therefore, deletion of E3 reduces the host immune response.

Advantages of Adenovirus Packaging

• Wide range of hosts
• High infection efficiency
• Large vector capacity
• Fast expression
• Low pathogenicity and high safety

Service Process

1) Construct an adenoviral vector expressing miRNA/shRNA and use PacI to digest and purify the plasmid to expose the ITRs.

2) Transfect the adenovirus expression clone into 293A cells. Cells were harvested to prepare virus crude extracts.

3) Infect 293A cells with the virus crude extract to amplify the virus. Determine the viral titer.

4) Add viral supernatant to target cells.

5) Analyse miRNA/shRNA expression and corresponding Knockdonw results.

Customer Provided

1) Plasmid expressing miRNA/shRNA (provide sequencing profile) or the name and sequence of the target gene to be Knockdown.

2) The required virus titer and total amount, and whether the virus needs to be purified.

3) If it is necessary to detect changes in the expression of the target gene, please provide the appropriate antibody.

Experiment Provided

1) Provide the recombinant adenovirus vector plasmid with PCR identification results.

2) Provide an adenovirus stock solution whose titer has been determined and can be directly used for subsequent analysis.