Abstract
BackgroundThe development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited.Principal FindingsWe established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments.Conclusions/SignificanceOur study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.
Highlights
High-throughput gene transduction methods are needed for gene function studies and drug discovery
The associated virus (AAV) plasmid, genomic structure of the helper virus used for recombinant AAVs (rAAVs) packaging, and electron microscopic assessment
AAV vectors harboring reporter genes were constructed by inserting the genes encoding Gaussia luciferase (Gluc) or enhanced green fluorescent protein (EGFP) between the cytomegalovirus (CMV) promoter and bovine growth hormone polyA (BGH polyA) in pAAV2neo (Fig. 1A) or pAAV5neo [14]. rAAVs were produced by infecting the AAV vector cell lines with recombinant herpes simplex virus carrying the AAV rep and cap genes (Fig. 1B); purification was achieved as described previously [15]
Summary
High-throughput gene transduction methods are needed for gene function studies and drug discovery. Different from conventional transfection or infection of living cells using DNA (or RNA) or a viral vector, reverse transfection or infection requires immobilization of the DNA (RNA) or viral vector on a solid support. Transduction is achieved by adding cells to the immobilized DNA or vector, which can save time and labor and reduce readout variation. The transfection conditions may need to be modified when different cell lines are used. We describe an RI protocol based on another frequently used viral vector, an adeno-associated virus (AAV). The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited
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