Construction of A Lentiviral Vector Simultaneously Expressing Active IGF1， EGF and FGF2

Abstract: Co-culture system and exogenous growth factors are commonly used in induction of directional differentiation of stem cells towards sensory hair cells. In order to combine these two strategies in further work， we designed a lentiviral vector which could simultaneously express active IGF1， EGF and FGF2. The coding sequence for active epidermal growth factor (EGF) was cloned by RT-PCR with RNA from human colorectal cancer SW620 cells， and the coding sequences for active insulin-like growth factor 1 (IGF1) and fibroblast growth factor 2 (FGF2) were artificially synthesized. The coding sequence for the signal peptide of human immunoglobulin (Igκ) responsible for protein secretion and sequence for internal ribosome entry site (IRES) responsible for translation initiation were cloned from plasmids pSecTag2A and pIRES2-EGFP， respectively. The coding sequences for Igκ and three growth factors were fused respectively with IRES spacers in between， cloned into a lentiviral expression plasmid with green fluorescent protein (GFP) reporter， pLVX-IRES-ZsGreen1， and packaged into recombinant lentiviruses. HEK293T cells were infected by the lentiviruses and screened for single cell clones with GFP expression by limited dilution. The expression of IGF1， EGF and FGF2 were examined by Western blot， and proliferation assay of human lung cancer A549 cells was used to evaluate the activity of the secreted growth factors. In conclusion， we successfully constructed a lentiviral vector which could simultaneously express functionally active IGF1， EGF and FGF2 when introduced into mammalian cells in this work， providing a powerful tool for designing engineered cells expressing these three growth factors used in co-culture system to induce directional differentiation of stem cells.

CSTR: 32200.14.cjcb.2014.11.0007