The Construction And Identification Of The Neuritin Transgenic Mice

Object: The aim of this paper is to identify the rat Neurtin gene, and rat-neuritin-overexpressing transgenic mice were obtained by pronuclear microinjection method, was constructed to provide experimental animal model for the study of Neuritin function after nerve injury at systems biology level, and to provide the experimental basis for further study the function of Neuritin gene. Methods:1) Comparative approaches were employed to design two primers which were homologous to the two transcripts of mouse Neuritin, and RT-PCR method was used to fish rat Neuritin transcripts. And bioinformatics methods were employed to predict analyze rat Neuritin sequence homology and its protein structure. 2) The eukaryotic expression vector with a super CMV promotor(pcDNA3.1-neuritin) was constructed: Firstly, The pair primers wih Xho- I and Nhe- I restriction sites were designed according to rat Nueritin cDNA reference sequences and the desired rat Neuritin coding region fragment was amplified. Secondly, the target PCR product and pcDNA3.1(+) blank plasmid were digested with Xho- I and Nhe- I digestion, and the two purified digestion products were ligated each other and then transformed into E. coli DH5 alpha, and the potive recombinant pcDNA3.1(+)-neuritin plasmids were identified by a serial methods(including colony PCR, double enzyme digestion and DNA sequencing). 3) Neuritin transgenic mice were obtained wih the method of zygote pronuclear microinjection: the recombinant plasmid was extracted after the postive colony, and the pcDNA3.1(+)-Neuritin plasmid was digested with Bgl II and StuⅠ, and a 2.3 Kb target fragment was purified and then microinjected into fertilized eggs, screening the living embryos and subsequently were transplanted into pseudo-pregnant recipient mice oviduct. The positive offsprings were identified by PCR screening method. Results: 1) The studies indicated that there is only one Neuritin transcript in rat, encoding 142 amino acids, which is highly conserved among species; Tertiary structure analysis shows that rat Neuritin is made up of six α-helix, and its structure and physicochemical properties is tantamount to the human Neuritin. 2)The eukaryotic expression vector pcDNA3.1(+)-Neuritin was successfully identified by Xho- I and Nhe- I double digestion and sequencing. 3) Transgenic mice establishment by pronuclear microinjection: the target transgenic fragments(3ng/μl) were microinjected into 711 fertilized eggs, and 275 eggs were survived after a short-term culture in vitro, the survival rate was 38.67%, and all these survival eggs were transplanted into 30 recipients, 26 pregnant transplant recipients produced a total of 45 offspring mice, three offspring transgenic mice were idenfied by using two different pairs of PCR primers which located into vector and Neuritin gene, and the integration rate was 6.66%. Breeding lines transgenic mice and subsequent analysis remains to be further study. Rt-pcr and sequencing identified four F1 rat neuritin gene expression in mice tail, positive transgenic expression rate is 28.57%. Conclusion: 1) Only one Neuritin transcript was identified in rat. 2) The pcDNA3.1(+)-Neuritin eukaryotic expression vector was successfully constructed; 3) Neuritin-overexpessing transgenic mice model was successfully established.