Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression

Zitzmann, Jan; Schreiber, Christine; Eichmann, Joel; Bilz, Roberto Otmar; Salzig, Denise; Weidner, Tobias; Czermak, Peter

doi:10.1016/j.btre.2018.e00272

Cited by 16 publications

(13 citation statements)

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“…1a–c ). The next step in metastasis—formation of colonies within the distant organ—involves clonal growth from a single cell 28 , which is dependent on factors secreted by other cells; in the laboratory, conditioned medium (CM) is generally used to generate clonal cell lines 29 – 32 . To assess the role of secreted factors on the colony-forming capacity of MDA.MB.231 cells, we measured the CM requirements for anchorage-dependent clonal growth (Fig.…”

Section: Resultsmentioning

confidence: 99%

ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells

et al. 2020

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Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.

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Section: Resultsmentioning

confidence: 99%

ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells

et al. 2020

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“…A similar strategy to combine GFP and BSD resistance was used to select insect cell lines for recombinant protein expression. 28 The GFP-BSD resistance fusion gene was also successfully used for selection of engineered recombinant poxviruses. 29…”

Section: Resultsmentioning

confidence: 99%

Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

Mei

Jiang

et al. 2019

Human Gene Therapy Methods

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Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for largescale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 10 5 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.

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“…Stable S2 transfectants were generated by adding hygromycin to a final concentration of 250 μg/mL every week for at least 4 weeks. Single cell clone expressing recombinant proteins was obtained via the limiting dilution method [ 34 ]. Recombinant proteins were secreted into culture supernatant after CuSO 4 (500 µM) induction and then purified using Profinity™ IMAC uncharged column (Bio-Rad, Hercules, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Targeting Haemagglutinin Antigen of Avian Influenza Virus to Chicken Immune Cell Receptors Dec205 and CD11c Induces Differential Immune-Potentiating Responses

et al. 2021

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Improving the immunogenicity and protective efficacy of vaccines is critical to reducing disease impacts. One strategy used to enhance the immunogenicity of vaccines is the selective delivery of protective antigens to the antigen presenting cells (APCs). In this study, we have developed a targeted antigen delivery vaccine (TADV) system by recombinantly fusing the ectodomain of hemagglutinin (HA) antigen of H9N2 influenza A virus to single chain fragment variable (scFv) antibodies specific for the receptors expressed on chicken APCs; Dec205 and CD11c. Vaccination of chickens with TADV containing recombinant H9HA Foldon-Dec205 scFv or H9HA Foldon-CD11c scFv proteins elicited faster (as early as day 6 post primary vaccination) and higher anti-H9HA IgM and IgY, haemagglutination inhibition, and virus neutralisation antibodies compared to the untargeted H9HA protein. Comparatively, CD11c scFv conjugated H9HA protein showed higher immunogenic potency compared to Dec205 scFv conjugated H9HA protein. The higher immune potentiating ability of CD11c scFv was also reflected in ex-vivo chicken splenocyte stimulation assay, whereby H9HA Foldon-CD11c scFv induced higher levels of cytokines (IFNγ, IL6, IL1β, and IL4) compared to H9HA Foldon-Dec205 scFv. Overall, the results conclude that TADV could be a better alternative to the currently available inactivated virus vaccines.

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Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression

Cited by 16 publications

References 50 publications

ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells

ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells

Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

Targeting Haemagglutinin Antigen of Avian Influenza Virus to Chicken Immune Cell Receptors Dec205 and CD11c Induces Differential Immune-Potentiating Responses

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