Fig 1.
Production of AAV6 vectors in HEK293T cells.
(A) The components of the pAAV donor plasmid and pDGM6 helper plasmid. The pAAV donor plasmid vector comprises ITR, left homology arm of AAVS1 gene (AAVS1-LHA), EF1α promoter, the gene of interest (GOI) tagged with mCherry, polyA tail, right homology arm of AAVS1 gene (AAVS1-RHA) and ITR. The pDGM6 helper plasmid consists of the AAV6 cap genes, the AAV2 rep genes, and the adenovirus helper genes. (B) Schematic of AAV6 production by co-transfection of the pAAV donor and pDGM6 helper plasmid vectors into HEK293T cells. (C) Fluorescence microscopic analysis of the untransfected and transfected HEK293T cells at 3 days post-transfection. Scale bar = 100 μm. (D) The qPCR standard curve was created by plotting the logarithmic DNA concentrations against Cq values.
Fig 2.
(A) Schematic diagram of the gene-editing strategy targeting the AAVS1 locus using the CRISPR/Cas9 RNP complex and the recombinant AAV6 vectors in human iPSCs. HR = Homologous Recombination. (B) Microscopic fluorescence analysis of human iPSCs shows mCherry expression on days 1, 4 and day 6 post-nucleofection from three different conditions. Scale bar = 100 μm (Day 1) and 200 μm (Days 4 and 6). (C) The percentage of mCherry+ cells on day 6 post-nucleofection as analyzed by flow cytometry.
Fig 3.
Clonal isolation and characterization of the genetically engineered human iPSCs.
(A) The mCherry expression of human iPSCs after limiting dilution. Scale bar = 200 μm. (B) Karyotype analysis by standard G banding demonstrated that the engineered human iPSCs exhibited normal karyotype (46, XX). (C) Immunofluorescence staining of pluripotent markers NANOG, OCT4, SSEA-4, TRA-1-60 and TRA-1-81. The nuclei were stained with DAPI. Scale bar = 100 μm. (D) PCR amplification of genomic DNA extracted from the wild-type iPSCs, genetically-engineered iPSCs, pAAV donor plasmid and non-template control (NTC). The major band indicates a successful transgene knock-in at the AAVS1 locus.