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Custom AAV production Introduction Recombinant AAV (rAAV) particles are powerful tools for biological and medical studies. A large number of published papers have been involved in using of AAV delivery. Since AAV infection rarely leading to serious immune response, it is more widely used in vivo for animal injections compared with in vitro cell infection. Although AAV packaging has been well investigated, it is time-consuming to obtain high quality AAV particles, especially for researchers with no experience. Wild type AAV has a genome size of ~4.7 kb which limits its packaging capacity. AAV titer is inversely proportional to the insert size between two ITRs. With the insert size more close to AAV genome size, it is harder to achieve high titer AAV particles. Experts at RGBiotech have many years of experience in the field of AAV. We know how to get the optimal AAV particles for each special project. We can start from the very beginning of experimental design, to sequence synthesis, vector construction, AAV production and purification. We can also start form AAV packaging if the customer could provide the AAV expression / transfer plasmid. So far, we have successfully helped hundreds and thousands of clients in their projects relating to AAV utilization. Features - One-stop AAV service: start from any step of AAV production- Provide multiple options of AAV serotypes for infection of different cell types: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-DJ, AAV-DJ8, AAV-PHP.B - Provide multiple options of promoters: promoterless, CMV, CAG, Synapsin, EF1α, UBC, CBh, TRE-miniCMV, CMVtight, ALB, MHCK7, ApoE/AAT1, CaMKII, ELA1, Enh358MCK, Ctnt, GFAP, MBP, SST, TBG, αMHC, hRPE, mIP1, tMCK, H1, U6 - Choice of AAV expression plasmid construction: one expression cassette for single expression, or two expression cassettes for independent expression - Insert types: mammalian genes (human/mouse/rat etc.), virus gene (e.g., HBV genome), shRNA/miRNA encoding sequence etc. - High titer: up to 1 x 10^13 GC/mL, even 1 x 10^14 GC/mL - High purification via 2 x CsCl ultra-centrifugation or Iodixanol gradient ultracentrifugation - Suitable for both in vivo animal experiments and in vitro cell experiments - Fast turnaround time: typical 2 ~ 3 weeks - Save time from AAV marketing research and provide competitive price in the market Service Workflow - Communication with customers to discuss the fit-to-project solution- Complete AAV production outline: -> sequence design and synthesis -> construction of AAV expression plasmid -> maxi-prep of AAV expression plasmid and the packaging plasmids -> 293 cell culture and co-transfection of the plasmids -> AAV purification via 2 x CsCl ultra-centrifugation or Iodixanol gradient ultra-centrifugation -> AAV titration via qPCR - Technical support for AAV using Case Studies 
Figure1. Fluorescent images of heart cryosections from mice injected with AAV1-GFP, AAV2-GFP, AAV6-GFP, AAV8-GFP, AAV9-GFP through jugular vein. Images were captured on day 20 after AAV administration. 
Figure2. Left) Bioluminescent images of mice injected with AAV1-luciferase, AAV2-luciferase, AAV6-luciferase, AAV8-luciferase, AAV9-luciferase through jugular vein. Images were captured on day 28 after AAV administration. Right) Luciferase activities of protein samples extracted from tissues (brain-B, kidney-K, spleen-S, thymus-T, intercostal muscle-Im, Gastrocnemius-Gn, liver-L, heart-H) on day 28 after AAV administration. |
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