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Small viruses that can carry relatively small payload, but they do not exhibit pathogenicity nor cytotoxicity in the host organism. They can mediate gene transfer to both mitotic and non-mitotic cells, they lack the risk of oncogene activation, since they can exist stably in an episomal state with a low rate of chromosomal integration. AA virions are small (20-25 nm diameter) and have no enveloped, they carry ssDNA and they are defective virus dependent on the presence of a helper virus for replication.
CNS, photoreceptor cells, LungAstrocytes, hippocampal neurons
CNS, photoreceptor cells, skeletal muscle, Heart, Liver, Pancreas
High specificity in Auditory Cortex
CNS, skeletal muscle, Heart, Liver, LungNeurons, hippocampus, auditory cortex
A) AAV are non-enveloped, icosahedral and small. They package ~4.7 kB, flanked by ITR singled stranded DNA containing two Open Reading Frames encoding for Replication (rep) and capsid (cap) proteins. B) We currently work with recombinant AAV, meaning that AAV ssDNA no longer contains the cap and rep genes but the transgene which will be introduced in the host cells. Therefore, production of these viruses is achieved by co-transfection of the transgene vector and an AAV helper plasmids that contains the necessary genes for the packaging and virus production within the host cells.
AAV pseudotyping: Define as the mix of a capsid and genome from different viral serotypes. We currently have in stock some plasmids for AAV production purchased from PasmidFactory including pDM and pDG (Wild type AAV2 serotype), and a series of pDP including rep2 (structural protein from AAV2 serotype) and different cap proteins (capsid protein) conferring different tropisms depending which cap proteins are coded.
Production and packaging available from different pseudotypes. Purchased from PlasmidFactory which holds the license from the German Cancer Research Center, we provide the user with several serotypes to choose from, depending on their needs:
The facility has acquired the necessary plasmids for production of the following pseudotypes:
These plasmids expand the current pseudotype library at the customers disposal. These pseudotypes are based on the AAV9 pseudotype that allows the production of high titer AAV viruses that allows a less invasive method of the CNS (PHP.eB) or PNS (PHP.S) cell infections by systemic inoculation of the virus via IP while still retaining their specificity to infect only Nervous system cells (Chan et al., 2017; Deverman et al., 2016).
Note: AAV vector plasmids are notorious for the instability and frequent deletion of the ITRs when propagated in E. coli. For this reason, it is important to carefully confirm the integrity of every preparation. Even the best plasmid preparations contain significant quantities (5–15%) of DNA containing small deletions in the ITR, which during cloning procedures can be randomly selected and amplified. The integrity of the ITRs is typically monitored by restriction enzyme analysis sufficient to identify small deletions (20 bp). Most commonly this is achieved with digestion with Sma I or Srf I, which cut twice within an unstable portion of the ITR. Because of these stability issues, most investigators utilize a recombination deficient E. coli strain, such as SURE (Agilent Technologies, ref#200152) or NEB stable (NEB, ref#C3040I) for AAV vector plasmid propagation and cloning.
~500 µl in chosen size aliquots, titer ≥1010 vg/mL (viral genomes/mL). Prices include titration by qPCR or FACS:
Physical titration: 10.000 SEK/virus for KI users; 12.000 SEK/virus for external users.
Functional titration (if available): 9.500 SEK/virus for KI users, 11.000 SEK/virus for external users.
The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression, are attributes that bring lentiviral vectors to the forefront of gene delivery vehicles for research.
The Gammaretrovirus is a sister genus to the lentivirus clade. Like the lentivirus, it is a member of the Orthoretrovirinae subfamily of the retrovirus family, they share common properties but the main difference between these two viruses is that Gammaretroviruses only infect dividing cells.
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