A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase. Tratschin JD, West MH, Sandbank T, Carter BJ. Midbrain dopamine cells do not show obvious signs of degeneration ( N, o) and there is only a mild inflammation response at the site of the injection as indicated by the microglial marker Ox42/cd11b ( P, q). ( M) Quantifications of TH-ir cells in the substantia nigra, expressed as percentage of the injected side compared to the uninjected side. The dotted red line indicates the threshold of well lesioned animals. ( K) The response to 2.5 mg/kg d-amphetamine was averaged over 90 min. Optical density analysis using ImageJ for the three proteins GFP, Ox42 and TH are presented in ( H– J, L), respectively. ( D– G) High power confocal microscopy images displaying the strong expression of GFP in the terminal fibres in the striatum ( D, F) and the cell bodies in the substantia nigra ( E, G). Note that overviews for the four remaining control groups are supplied in the Supplementary Fig.
#AWAVE VS CHICKENSYS VS CDXTRACT SERIES#
Overview of a coronal 1:12 series of the brains labelled for GFP for the two main experimental groups ( A-Iod-GFP B-Chl-GFP) as well for the PBS-injected control group ( C). In vivo comparison of production methods on transgene expression and inflammation. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain.
Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment.
In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases.
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