Screening Gene Mutation Of Familial Alzheimer's Disease And Construction Of APP Expression Vectors

Purpose:Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly population. Clinical and research data show that early-onset of familial AD is associated with three genetic factors so far including presenilin 1 gene (PS-1), presenilin 2 gene (PS-2) and amyloid precursor protein (APP) gene. Most of FAD were reported from foreign researchers, while very few of them were reported in Chinese people. So, we screened mutations in the three genes in some Chinese FAD families identified by clinical diagnosis, aiming to know AD related gene mutation frequency and hot spot in China. As for novel mutation identified, further research would be completed to understand underlying pathogenic mechanisms.Methods:Collect peripheral blood from AD families and extract genome DNA. Design primers which could amplify encoding exons of PS-1 and PS-2, and exonl6 and 17 of APP. Then PCR products were sequenced. A novel mutation which had been screened was introduced into APP695 by Megaprimer-PCR, and eukaryotic expression vectors of APP695 wild-type and mutation genes were constructed. Resultant plasmids were transfected into HEK293 to establish stable expression cell lines. Meanwhile, we constructed recombinant adenovirus plasmids of the wild-type and mutation genes, generated recombinant adenoviruses in HEK293 packaging cells, identified recombinant adenoviruses by Western Blot assay.Result:We identified a mutation in PS-1 (R278I) and a novel mutation in APP (K724X), and constructed eukaryotic expression vectors pcDNA3.1(-)-APP695WT, pcDNA3.1(-)-APP695K724X and pcDNA3.1(-)-APP695K724N. The stable expression cells were also established. Besides, we constructed recombinant adenovirus plasmids pAd-APP695WT, pAd-APP695K724X and pAd-APP695K724N, and generated recombinant adenoviruses successfully identified by Western Blot assays respectively.Conclusion:A mutation in PS-1 (R278I) was screened and a novel APP gene mutation (APPK724X) causing FAD was identified in a Chinese family. Eukaryotic expression vectors of APP695 were constructed and stable cell lines were established. Also, recombinant adenovirus plasmids were constructed and recombinant adenoviruses were successfully packaged.