Cloning and identification of normal mouse NT3 gene and the establishment and screening of cell strain expressing high level of NT3 gene / 第三军医大学学报

ABSTRACT

Objective To clone and identify neurotrophic factor NT3 gene from mouse liver so as to establish a cell strain expressing high level of NT3 gene. Methods The target genes amplified by RT PCR were cloned into the shuttle vector pExchange 1neo, and then the DNA sequence was analyzed by enzyme digestion and sequencing. The recombinant expression vector pExchange NT3 1neo was employed to transfect the embryonic stem cell strain MESPU35, and then the transfected cells were selected by G418. The NT3 level in the transfected cells was detected by RT PCR. Results A gene fragment of 777 bp was obtained by RT PCR, and the DNA sequence was identical to mice NT 3 gene sequence of GenBank. The recombinant vector was constructed successfully and the constructed cell strain could express high level of NT 3 gene. Conclusion The successful cloning of NT3 gene from mouse liver, construction of pExchange NT3 1neo expression vector, and establishment of cell strain stably expressing high level of NT3 gene have laid a foundation for the further studies.