Limits...
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.

Show MeSH

Related in: MedlinePlus

Response to NaB. Black bar, without NaB; gray bar, with NaB.A. AAV1 RI-based assay. B. AAV2 RI-based assay.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2957432&req=5

pone-0013479-g004: Response to NaB. Black bar, without NaB; gray bar, with NaB.A. AAV1 RI-based assay. B. AAV2 RI-based assay.

Mentions: We next evaluated the responses of the rAAV-transduced cell lines to NaB, which was used as an interfering factor. NaB is known to enhance gene expression [16]. Cells were applied to wells coated with rAAV1 or rAAV2 in the presence or absence of 10 mM NaB. Then, 24 h later, Gluc activity in the supernatant was measured. Gluc activity in the cells transduced by rAAV1 (Fig. 4A) or rAAV2 (Fig. 4B) was increased significantly, especially for HEK293 and BHK21. Of rAAV2, Gluc activity in BHK21 increased from 3×105 to 1.6×106 relative light units (RLU; Fig. 4B). Of rAAV1, Gluc activity in the BHK21 cells increased, from 1.5×105 to 4×105 RLU (Fig. 4A). In conclusion, our data show that our system can be used to monitor the response to a modulating factor with substantial sensitivity.


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)

Response to NaB. Black bar, without NaB; gray bar, with NaB.A. AAV1 RI-based assay. B. AAV2 RI-based assay.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013479-g004: Response to NaB. Black bar, without NaB; gray bar, with NaB.A. AAV1 RI-based assay. B. AAV2 RI-based assay.
Mentions: We next evaluated the responses of the rAAV-transduced cell lines to NaB, which was used as an interfering factor. NaB is known to enhance gene expression [16]. Cells were applied to wells coated with rAAV1 or rAAV2 in the presence or absence of 10 mM NaB. Then, 24 h later, Gluc activity in the supernatant was measured. Gluc activity in the cells transduced by rAAV1 (Fig. 4A) or rAAV2 (Fig. 4B) was increased significantly, especially for HEK293 and BHK21. Of rAAV2, Gluc activity in BHK21 increased from 3×105 to 1.6×106 relative light units (RLU; Fig. 4B). Of rAAV1, Gluc activity in the BHK21 cells increased, from 1.5×105 to 4×105 RLU (Fig. 4A). In conclusion, our data show that our system can be used to monitor the response to a modulating factor with substantial sensitivity.

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