In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

摘要

BACKGROUND:Neurotrophin-4(NT-4) can promote neuronal growth,development, differentiation,maturation,and survival.NT-4 can also improve recovery and regeneration of injured neurons,but cannot pass through the blood-brain barrier,which limits its activity in the central nervous system.Delivering NT-4 into the central nervous system via cells or vectors may have therapeutic benefit. OBJECTIVE:To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-N1. DESIGN,TIME AND SETTING:Neural genetic engineering experiment.The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008. MATERIALS:The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences;embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College.TRIzol RNA extraction Kit was purchased from Sigma (USA),One Step RNA PCR Kit(AMV) etc.were from Takara(Dalian,China). METHODS:Total RNA was extracted from human embryonic brain tissues using Trizol.The agarose gel electrophoresis showed two bands:18 S and 28 S,which were essential subunits of total RNA.The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction. MAIN OUTCOME MEASURES:The sequencing results of the DNA in the recombinant of NT-4- pEGFP-N1. RESULTS:The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight. CONCLUSION:The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.