Establishment of an AAV Reverse Infection-Based Array

Background: 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. Principal Findings: We 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 vectormediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance: Our study provides a novel method for establishing a highly efficient gene transduction arra

High-Throughput Functional MicroRNAs Profiling by Recombinant AAV-Based MicroRNA Sensor Arrays

BACKGROUND: microRNAs (miRNAs) are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking. PRINCIPAL FINDINGS: We describe an adeno-associated virus (AAV) vector-based microRNA (miRNA) sensor (Asensor) array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia luciferase (Gluc) and a firefly luciferase (Fluc) expression cassette to sense functional miRNA and to serve as an internal control respectively. Using this array, we acquired functional profiles of 115 miRNAs for 12 cell lines and found "functional miRNA signatures" for several specific cell lines. The activities of specific miRNAs including the let-7 family, miR-17-92 cluster, miR-221, and miR-222 in HEK 293 cells were compared with their expression levels determined by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). We also demonstrate two other practical applications of the array, including a comparison of the miRNA activity between HEK293 and HEK293T cells and the ability to monitor miRNA activity changes in K562 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). CONCLUSIONS/SIGNIFICANCE: Our approach has potential applications in the identification of cell types, the characterization of biological and pathological processes, and the evaluation of responses to interventions

Optimization of rAAV RI.

<p>A. Transduction efficiency of different quantities of rAAV2-Gluc using 4×10⁴ BHK21 cells; the transduction efficiency is represented by Gluc activity. B. Optimization of the virus/cell ratio. Different numbers of BHK21 cells were applied to a 96-well plate pre-coated with 5×10⁸ viral genomes/well. The transduction efficiency is represented by the EGFP intensity. C. Temperature stability assessment of rAAV2-Gluc. In total, 5×10⁸ viral genomes of rAAV were applied to each well. The plates were then treated at 4, 37, 42, or 56°C for 24 h, followed by the application of 4×10⁴ BHK21 cells per well. Gluc activity was measured 24 h later.</p

Response to NaB. Black bar, without NaB; gray bar, with NaB.

<p>A. AAV1 RI-based assay. B. AAV2 RI-based assay.</p

miRNA activity profiles for the twelve cell lines.

<p>(A) Activities of 115 miRNAs in 12 cell lines were detected. (B–G) Comparison of specific miRNA activities in 12 cell lines. miR-199a-3p activity was specifically high in BJ, C2C12, and BHK21 cells (B). miR-143 activity was specifically high in BJ and C2C12 cells (C). miR-21 activity was high in all of the cell lines excluding HEK293T (D). miR-221/222 cluster activities were high in all cell lines excluding U937 and K562 (E). miR-142-3p and miR-142-5p activities were specifically high in K562 and U937 cell lines (F). miR-122 activity was specifically high in Huh7/CD81, while miR-194 activity was high in both Huh7/CD81 and HepG2 cells (G). RIF, relative inhibiting fold. Error bars correspond to mean±SD (n = 3).</p

Comparison of miRNA activity profiles between HEK293 and HEK293T cells.

<p>(A) miRNA activity profiles for HEK293 and HEK293T were established using the miRNA Asensor arrays. (B) Several miRNAs were selected and their activities compared. Compared to HEK293T cells, miR-17-5p, miR-18a, miR-20a, and miR-106a activities were lower in HEK293 cells, while miR-21, miR-222, let-7a, let-7b, let-7c, let-7e, and let-7f were higher. (C) miR-21 expression level was detected by QRT-PCR. The miR-21 activity was consistent with its expression level in the two cell lines. 2^ΔCt was used to indicate the miRNA expression levels. ΔCt = Ct_U6−Ct_miRNA. RIF, relative inhibiting fold. Error bars correspond to mean±SD (n = 3). Student T test was used for statistical anylasis. *indicates <i>P</i> values<0.05 and ** <i>P</i> values<0.01.</p

Comparison of miRNA activities with expression levels in HEK293 cells.

<p>(A, D) The activities of members of the let-7 family were not consistent with their expression levels. (B, E) miR-221 and miR222 activities were consistent with their expression levels. (C, F) The consistency of miRNA activity with expression levels was complex in the miR-17-92 cluster. For miR-18a, the miRNA activity was not consistent with its expression level. For miR-17-3p, miR-17-5p, miR-19a, miR-19b, miR-20a and miR-92a, the miRNA activities were consistent with their expression levels. 2^ΔCt was used to indicate miRNA expression. ΔCt = Ct_U6−Ct_miRNA. RIF, relative inhibiting fold. Error bars correspond to mean±SD (n = 3).</p

Changes in miRNA activity induced by TPA in K562 cells.

<p>(A) Morphological changes in K562 cells induced by TPA. K562 cells were cultured in DMEM (10% FBS) containing 16 nM TPA. Twenty-four hours later, the morphology of K562 cells was altered. (B) Comparison of miRNA activity induced by TPA versus untreated K562 cells. The miRNA activity profiles with or without TPA were detected using miRNA Asensor arrays and miRNA activities with obvious changes are shown. Error bars correspond to mean±SD (n = 3). Student T test was used for statistical anylasis. * <i>P</i> values<0.05; ** <i>P</i> values<0.01.</p