Design and Production of AAV Vectors

Adeno-associated virus vector production was enabled by the molecular cloning of double-strand AAV DNA into bacterial plasmids followed by transfection into helper virus-infected mammalian cells.[1,6] This results in replication of the AAV genome free of any plasmid sequence to yield AAV particles. For AAV vector construction, the cis-acting ITR sequences must be retained and the unique sequence is replaced with foreign DNA.[1,6] There is a limit of about 5 kb of DNA that can be packaged in an AAV vector particle. Production of AAV vectors in host cells can be accomplished entirely by DNA transfection-based methods or by cell-based systems that do not require DNA transfection.[1,6] Both approaches can give a specific productivity in excess of 104 vector particles per cell, but the cell-based systems may be more amenable to scale-up for commercial production.

In DNA transfection-based systems, human 293 cells are transfected with DNA plasmids containing the AAV vector cassette, the rep and cap genes, and the adenovirus E2A, E4, and VA genes. Alternatively, stable producer cell lines that contain the rep and cap complementing genes and the vector genome are infected with Ad. Producer cell lines are readily scalable AAV, but a new producer cell line must be generated for each individual AAV vector. A third approach uses a packaging cell line containing a rep-cap gene cassette that is then coinfected with an Ad/AAV hybrid virus, which is an E1 gene-deleted Ad containing the AAV-ITR vector cassette, and Ad to provide E1. After infection, the rep-cap genes, the AAV-ITR cassette is amplified and packaged into AAV particles. The same packaging cell line can be used for production of different AAV vectors simply by changing the Ad/AAV hybrid virus. Variations of these methods use herpes simplex virus (HSV) in the production of AAV vectors by utilizing a hybrid virus in which the AAV rep-cap genes were inserted into the HSV genome. Insect cells may be used with baculovirus vectors containing rep-cap gene cassettes or the AAV-ITR vector cassette to produce AAV vectors. Adeno-associated virus vector purification is generally accomplished by chromatographic methods, including ion exchange and affinity resins.[1] Adeno-associated virus vector amounts and concentrations are measured by the number of vector genomes that are encapsidated, and thus protected from digestion by DNAse, and are expressed in units of DNAse-resistant particles (DRP).

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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