Establishment Of A HEK293 Cell Line Stably Expressing Human APP695 Gene

Objectives: Alzheimer's disease (AD) is a widespread, neurode- generative disorder of the eldly. This disease is characterized pathologically by senile plaques in the cerebral cortex and the hippocampus containing the amyloidβ-pepide(Aβ), albeit largely outside the brain parenchyma. The accumulation and deposition of Aβin the brain over decades leads to neuron dysfunction and eventually clinical manifestation of the disease. Recent research demonstrates that Aβis 40 to 42 amino acids in length and is generated by proteolytic cleavage of the much larger amyloid precursor protein(APP).Current research demonstrates that there is an increasing expression of APP in the diseased-damaged brain. The transcripts of APP ranging in predicted size from 695 to 770 aa. APP695 is preferentially expressed in neuronal tissue, leading to the speculation that production of Aβ. The familial form of Alzheimer's disease may be caused by APP mutations, which increase Aβproduction or lead to an increased proportion of Aβending at residue 42. A pathogenic mutation at codons 670/671 in APP(APP"Swedish") leads to enhanced cleavage at theβ-secretase scissile bond and increased Aβformation. Mutations in the APP gene account for only a minority of familial AD cases. In this study, the eukaryotic expression of the Human APP695sw and APP695wt was established to provide a model for researching Alzheimer's disease.Methods: The plasmid containing APP695sw/peak12 , APP695wt /peak12 and peak12 were introduced into NovaBlue E coli. Then, the plasmid was extracted through minibest plasmid purification kit after transformation. Cultured human embryonic kidney epithelial cells (HEK293) were transfected by LIPOFECTAMINE 2000. The HEK293 cells which stably expressed the APP of human would be survived in the further culture medium containing puromycin antibiotic. Last, the transfection result was demonstrated by immunocytochemistry.Result: The result showed that the specific value of plasmid was larger than 1.8 and less than 2.0. The density and purity can satisfied test requirement. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics at 37℃and 5% CO2. The cell population and quality can satisfied test requirement after high passage. The sensitivity test of puromycin defined the least concentration that resists cell growth. A cell clone appeared 16 days after screening. The level of APPsw increased in APPsw over-expressing cells with the methods of immunohistochemistry staining, but the level of APPwt had no remarkable difference.Conclusions: The plasmid APP695sw/peak12 had been introduced into HEK293 cells. Immunohistochemistry staining indicated that the level of APP695sw was increased in APP over-expressing cells.