Establishment of an Engineered Cell Secreting Multiple Types of Active Factors
ZHOU Menglei1, YANG Beibei1*, CAO Jiang2*
The purpose of this work is to establish an engineered cell which secrets multiple types of active factors, and can be used in co-culture for the induction of stem cell differentiation towards cochlear neuron-like cells. The coding sequences of mature peptides of rat BDNF (brain-derived neurotrophic factor) and NT3 (neurotrophin 3) were linked by IRES (internal ribosome entry site), inserted into lentiviral expression vector pLVXIRES-tdTomato and packaged into lentiviral particles, and used to infect HEK293T cells and HEK293T/3GF cells (which can express active form of EGF (epidermal growth factor), IGF1 (insulin-like growth factor 1) and FGF2 (fibroblast growth factor 2). Stable transfectants were cloned by limited dilution. Western blot was used to examine the expression of BDNF and NT3 in culture supernatant. The proliferation of human lung cancer A549 cells and rat pheochromocytoma PC-12 cells, as well as the differentiation status of PC-12 cells were used to assess the activity of the secreted factors. The results showed that the engineered cell expressing secretory active form of EGF, IGF1, FGF2, BDNF and NT3 was successfully established, which provides a powerful tool for the subsequent co-culture with stem cells.
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