Isolation of Full-length Rat and Mouse Amyloid Precursor Protein (App) cDNA from Glioma Cell Line by Two-step PCR Method

Abstract

Full-length cDNA of rat and mouse beta-amyloid precursor protein gene (App) were isolated from glioma cell line (NG-108-15). Because of the high molecular weight of the gene (around 2.2 kb), after the first stand of App cDNA was acquired by RT/PCR, the two-step PCR method was subsequently applied. The first PCR was conducted to fortify and generate two separated fragments (around 1.3 kb) of 5' and 3' ends (with partial overlapping, 0.41 kb) of App cDNA. The second PCR was carried out to obtain the full-length of App cDNA, which afterward was introduced into a mammalian vector, pcDNA3.1/Zeo+. Since NG108-15 cell is hybridoma line and the two-step method was applied in this study; therefore, four chimerical App cDNA clones were gained. Analysis of restrictive endonuclease enzyme fingerprints was subsequently exploited to identify chimera, rat and mouse App cDNA positive clones. By using the RT/PCR together with the two-step PCR technique in the present study is therefore shown to be useful and applicable method for isolating complete rat and mouse App genes from NG108-15 cell line, and possibly further applied for isolating other genes with high molecular weight and low levels of mRNA expression. The App cDNA obtained in this experiment can be utilized for upcoming experiments such as studying the mechanism of beta amyloid processing, its biological and clinical significance, and screening of new potential Alzheimer’s disease therapeutic agents.