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Establishment of an AAV reverse infection-based array.

Dong X, Tian W, Wang G, Dong Z, Shen W, Zheng G, Wu X, Xue J, Wang Y, Chen J - PLoS ONE (2010)

Bottom Line: 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.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.Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, China.

ABSTRACT

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 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/significance: Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.

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Related in: MedlinePlus

Optimization of rAAV RI.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.
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pone-0013479-g002: Optimization of rAAV RI.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.

Mentions: To determine the optimal amount of rAAV per well, 5×104, 5×105, 5×106, 5×107, and 5×108 viral genomes of rAAV2-Gluc were applied to each well. After drying, 4×104 BHK21 cells were applied to each well. Then, 24 h later, Gluc activity was measured. As shown in Figure 2A, Gluc activity was positively correlated with viral load; as the viral load increased, more Gluc activity was observed. Considering the different infectivity of AAV for different cells, we chose to coat each well with 5×108 viral genomes in each of the remaining experiments.


Establishment of an AAV reverse infection-based array.

Dong X, Tian W, Wang G, Dong Z, Shen W, Zheng G, Wu X, Xue J, Wang Y, Chen J - PLoS ONE (2010)

Optimization of rAAV RI.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957432&req=5

pone-0013479-g002: Optimization of rAAV RI.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.
Mentions: To determine the optimal amount of rAAV per well, 5×104, 5×105, 5×106, 5×107, and 5×108 viral genomes of rAAV2-Gluc were applied to each well. After drying, 4×104 BHK21 cells were applied to each well. Then, 24 h later, Gluc activity was measured. As shown in Figure 2A, Gluc activity was positively correlated with viral load; as the viral load increased, more Gluc activity was observed. Considering the different infectivity of AAV for different cells, we chose to coat each well with 5×108 viral genomes in each of the remaining experiments.

Bottom Line: 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.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.Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, China.

ABSTRACT

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 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/significance: Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.

Show MeSH
Related in: MedlinePlus