人胎脑神经生长因子4基因与质粒载体重组体的体外构建

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 v/a cells or vectors may have therapeutic benefit. OBJECTIVE： To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-NI. 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-NI. 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.

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

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.