An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.
Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.
Department of Pharmacology, Peking University, Health Science Center, Beijing, China.
Xiaojing
X
Zhang
Hui
H
Li
Yiqing
Y
Mao
Zhixin
Z
Li
Rong
R
Wang
Tingting
T
Guo
Ling
L
Jin
Rongjing
R
Song
Wei
W
Xu
Na
N
Zhou
Yizhuang
Y
Zhang
Ruobi
R
Hu
Xi
X
Wang
Huakang
H
Huang
Zhen
Z
Lei
Gang
G
Niu
David M
DM
Irwin
Huanran
H
Tan
D000544:Alzheimer Disease / Q000188:drug therapy*; D016229:Amyloid beta-Peptides / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017209:Apoptosis; D041702:BALB 3T3 Cells; D001483:Base Sequence; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000235:genetics*; D000077265:Donepezil; D004353:Drug Evaluation, Preclinical; D005702:Galantamine / Q000494:pharmacology; D015870:Gene Expression; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D007052:Ibuprofen / Q000494:pharmacology; D007189:Indans / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D010880:Piperidines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016335:Zinc Fingers
D016229:Amyloid beta-Peptides; D000894:Anti-Inflammatory Agents, Non-Steroidal; D002800:Cholinesterase Inhibitors; D007189:Indans; D010446:Peptide Fragments; D010880:Piperidines; D011994:Recombinant Proteins; C075222:amyloid beta-protein (1-42); D005702:Galantamine; D049452:Green Fluorescent Proteins; D000077265:Donepezil; D003851:Deoxyribonucleases; D007052:Ibuprofen