Paav dj

To obtain an AAVV expressing miR-199a-3p, all the plasmids required for AAVV generation (pAAV-IRES-GFP, pAAV-DJ, and pHelper) were purchased from Cell Biolabs (San Diego, CA, USA). The cassette expressing the miR-199a-3p sequence was obtained from the pIRES-miR-199a plasmid⁵⁰ (link) and cloned upstream of the IRES-GFP sequence into pAAV-IRES-GFP using XbaI sites. The infectious recombinant AAVV-199 was generated by a 1:1:1 (molar ratio) triple transfection of pAAV-199 with helper plasmids (pAAV-DJ and pHelper) into 293FT cells. Viral production and titration were performed as previously reported.⁵¹ (link)

AAV production was performed as previously described.²⁹ (link) In brief, an AAV vector plasmid (pAAV-CAG-mCherry, pAAV-CAG-Kitl or pAAV-CAG-Cre), an adenovirus helper plasmid (pHelper; Agilent Technologies, Santa Clara, CA), and an AAV helper plasmid (pAAV-RC; Agilent Technologies, pAAV1, pAAV6.2, pAAV9; gift from Penn Vector Core [University of Pennsylvania, PA], pAAV-DJ, pAAV-DJ8; Cell biolabs, San Diego, CA, and pAAV-7M8; Addgene, Boston, MA) were transiently transfected into 293T cells. Viral titers (in vector genomes (VG)/mL) were determined by real-time PCR using FastStart Universal SYBR Green Master Mix (Roche Diagnostic GmbH, Penzberg, Germany) and specific primers, as described previously (Watanabe et al., 2018). For screening, the titer of the virus was 1 × 10¹² vector genomes/mL. In some experiments, we also used AxCAN-Egfp (RIKEN BRC), CSII-EF1-IRES-Venus (RIKEN BRC) and CSII-EF1-Kitl. The titer of AxCAN-Egfp was 2.0 × 10⁸ plaque-forming units/mL. The titers of CSII-EF1-IRES-Venus and CSII-EF1-Kitl were 1.5 × 10⁹ transducing units (TU)/mL and 2.8 × 10⁹ TU/mL, respectively.

AAVs carrying hGFAP::Cre and CAG::FLEx-GFP for serotype testing in human islets were as previously described.⁵⁸ (link) AAV8-U6-mINS2utr5sg-EGFP-2cut (6.15 × 10¹³ GC/mL) was produced and purified by Penn Vector Core. AAV-DJ-U6-mINS2utr5sg-EGFP-2cut (2.92 × 10¹² GC/mL), AAV-DJ-U6-hINSin1sg-CopGFP-2cut (1.83 × 10¹³ GC/mL), AAV-DJ-U6-hINSin1sg-CopGFP-1cut (6.02 × 10¹² GC/mL), and AAV-DJ-nEF-Cas9 (3.83 × 10¹² GC/mL) were produced and purified as described below. Briefly, recombinant AAVs were produced in 293 AAV cells (Cell Biolabs, San Diego, CA, USA). Polyethylenimine (PEI, linear, MW 25,000) was used for transfection of three plasmids: the pAAV vector constructs, pAAV2/8-RC (Penn Vector Core) or pAAV-DJ (Cell Biolabs), and pHelper (Cell Biolabs). At 72 h post-transfection, cells were scrapped in their medium, centrifuged, and then frozen and thawed four times by placing alternately in dry ice-ethanol and a 37°C water bath to lyse the cells and release the virus. The resulting AAV crude lysate was purified by centrifugation at 54,000 rpm for 1 h in discontinuous iodixanol gradients with a Beckman SW55Ti rotor. The virus-containing layer was extracted, and viruses were concentrated by Millipore Amicon Ultra Centrifugal Filters (Millipore-Sigma, Bedford MA, USA). Virus titers were determined by quantitative PCR (qPCR) according to Addgene protocol.