Construction and identification of lentiviral vectors carrying the connexin43 gene

Abstract

Objective To construct and identify lentiviral vectors carrying the connexin43(CX43) gene. Methods Plasmids containing the CX43 gene and lentiviral vectors were digested using EcoRI/XbaI restriction enzymes, and the target gene fragments were cloned into the lentival vectors to result in CX43 recombinant lentiviral vectors (pHBLV-CMVIE-IRES-ZsGreen-CX43). CX43 recombinant lentiviral vectors were identified by restriction enzyme digestion and electrophoresis, and sequencing was carried on only for correct vectors after identification. The successfully-constructed CX43 recombinant lentiviral vectors and packaging plasmids were mixed and contransfected into 293FT cell for packaging and producting virus. Then the virus was collecting, concentrated and titrated in 293FT cells. Finally, the expression of CX43 gene was assessed by real-time polymerase chain reaction(PCR). Results The restriction enzyme digestion and electrophoresis and sequencing results proved that CX43 recombinant lentiviral vectors were constructed correctly. Lentiviral concentrated virus suspension titer was 1× 108/ml. The up-regulation expression of CX43 was detected correctly by real-time PCR in 293FT cells. Conclusions Stable lentiviral vectors expressing CX43 gene is constructed successfully. Key words: Connexin 43; Lentiviral vectors; Bone marrow