Establishment of APP and PS-1 double gene stably transfected cell lines / 中国药理学通报

ABSTRACT

AIM To study pathogenesis of Alzheimers disease and screen ?- secretase and ?- secretase inhibiting drug, PS-1(M 146L)and APP 751 gene double stably transfected CHO cell lines have been established. METHODS A? cDNA encoding human PS-1 was obtained by polymerase chain reaction from a human placental library. Mutant PS-1 (M 146L) cDNA was also generated by polymerase chain reaction from wild type PS-1 cDNA. Wild type and mutant PS1 were subcloned into CMV-based mammalian expression vectors PCI-neo,then recombined plasmid were co-transfected into APP expressing CHO cells at 1∶10 ratio using LipofectAmine. Stable expressing cell lines were screened and selected by using selection media. RESULTS Several CHO cell lines stably transfected with wide type or mutant PS-1 (M 146L) as well as APP 751 genes were established. Overexpression of PS-1 in CHO cell accumulated full length 45 kDa PS-1 protein. A? released to conditioned media were not changed in wild type PS-1 transfected APP expression CHO cells. A? 1～42 level in conditioned media of M 146L mutant PS-1 stably transfected of APP expression CHO cells were elevated about 1.6 fold. CONCLUSION Expression of M 146L mutant PS-1 in stably transfected APP expression CHO cells increased a secretion of A? 1～42. The PS-1 and APP double stably transfected CHO cell lines we generated can be used for either ?-or ?-secretase inhibitor study and related drug screening.