| Revista: | Brazilian Journal of Pharmaceutical Sciences |
| Base de datos: | PERIÓDICA |
| Número de sistema: | 000451949 |
| ISSN: | 1984-8250 |
| Autors: | Raigani, Mozhgan1 Barkhordari, Farzaneh1 Moazzami, Reza1 Davami, Fatemeh1 Mahboudi, Fereidoun1 |
| Institucions: | 1Pasteur Institute of Iran, Biotechnology Research Center, Teherán. Irán |
| Any: | 2022 |
| Volum: | 58 |
| País: | Brasil |
| Idioma: | Inglés |
| Tipo de documento: | Artículo |
| Enfoque: | Experimental, aplicado |
| Resumen en inglés | The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS |
| Disciplines | Biología |
| Paraules clau: | Biología celular, Bioquímica, Biotecnología, Líneas celulares, Selección celular activada por fluorescencia, Activación celular, Plasminógeno tisular |
| Keyword: | Cell biology, Biochemistry, Biotechnology, Cell lines, Fluorescence-activated cell sorting, Cell activation, Tissue plasminogen |
| Text complet: | Texto completo (Ver HTML) Texto completo (Ver PDF) |
