plenticmv tetr blast vector  (Addgene inc)

Journal: Biotechnology journal

Article Title: Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

doi: 10.1002/biot.202400104

Figure Lengend Snippet: FIGURE 2 Development of doxycycline-inducible 293-AST-TetR cell lines. (A) The relative mRNA expression of AST factors in 293-AST-TetR cells. (B) Proportion of suspended 293-AST-TetR cells upon incubation with various concentrations of doxycycline as analyzed via the CellTiter Glo Luminescent assay. Data are presented as means ± SEM. ****p < 0.0001 compared with the control cells. (C) Morphology of 293-AST-TetR cells treated with different doses of doxycycline for 48 h. (D) Image of 293-AST-TetR cells after treatment with 20 µg mL-1 doxycycline for 24, 48, and 72 h.

Article Snippet: We used the TetR plasmid (Addgene, 17492) to infect 293-AST cells and generate doxycyclineinducible 293-AST-TetR cells.

Techniques: Expressing, Incubation, Luminescence Assay, Control

Journal: Biotechnology journal

Article Title: Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

doi: 10.1002/biot.202400104

Figure Lengend Snippet: FIGURE 3 Inducible and reversible reprogramming of anchorage independency in 293-AST-TetR cells. Morphological observations of cells at different time points after the addition or withdrawal of doxycycline. (A) Control cells (mock). (B) 293-IKZF1-TetR cells. (C) 293-KLF1-TetR cells. (D) 293-BTG2-TetR cells.

Article Snippet: We used the TetR plasmid (Addgene, 17492) to infect 293-AST cells and generate doxycyclineinducible 293-AST-TetR cells.

Techniques: Control

Journal: Biotechnology journal

Article Title: Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

doi: 10.1002/biot.202400104

Figure Lengend Snippet: FIGURE 4 Improvement of the transfection efficiency in 293-AST-TetR cell lines. (A) Representative fluorescence images of inducible 293-AST-TetR cells transfected (1) in an adherent state or (2) in a suspended state after transfection during the adherent state, or (3) in stable 293-AST cells in a suspended state. (B), (C) Western blotting results demonstrating the transfection efficiency of inducible 293-AST-TetR cells compared to stable 293-AST cells. (D), (E) Immunoblots for EPO and IL-6 expressed in either inducible 293-AST-TetR cells treated with brefeldin A(BFA) for 3 h and 20 µg mL-1 doxycycline after a 24-h transfection or in stable 293-AST cells.

Article Snippet: We used the TetR plasmid (Addgene, 17492) to infect 293-AST cells and generate doxycyclineinducible 293-AST-TetR cells.

Techniques: Transfection, Fluorescence, Western Blot

Journal: Biotechnology journal

Article Title: Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

doi: 10.1002/biot.202400104

Figure Lengend Snippet: FIGURE 5 Establishment of AST-TetR cell lines with enhanced expression rate of recombinant proteins. (A) Schematic showing the method used for generating stable cell lines from 293-F, 293-AST, and 293-AST-TetR cells using mammalian expression vectors. (B) The viability of clones stably expressing EPO was measured during the selection process using the Cell Titer-Glo Luminescence reagent. (C), (D) Western blot analysis showing an increase in recombinant protein expression from 293-AST-TetR clones stably expressing EPO or IL-6. (E) Immunoblotting of p-STAT3 from Raw264.7 macrophage cells co-cultured with AST-TetR cells stably expressing IL-6.

Article Snippet: We used the TetR plasmid (Addgene, 17492) to infect 293-AST cells and generate doxycyclineinducible 293-AST-TetR cells.

Techniques: Expressing, Recombinant, Stable Transfection, Clone Assay, Selection, Western Blot, Cell Culture

Journal: Biotechnology journal

Article Title: Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

doi: 10.1002/biot.202400104

Figure Lengend Snippet: FIGURE 6 Schematic overview of reprogramming anchorage dependency via AST to develop cell lines for recombinant protein expression. (A) The procedure for generating 293-AST and 293-AST-TetR cell lines. (B) Increased transfection efficiency and recombinant protein production observed in adherent versus suspension state transfection. (C) The advantages of ASTExpress platform using inducible and reversible anchorage reprogramming for therapeutic protein expression.

Article Snippet: We used the TetR plasmid (Addgene, 17492) to infect 293-AST cells and generate doxycyclineinducible 293-AST-TetR cells.

Techniques: Recombinant, Expressing, Transfection, Suspension