Abstract
There are now various theories on the pathophysiology of Alzheimer’s disease (AD), the β-Amyloid (Aβ) cascade hypothesis has garnered a lot of interest. This text applied lentivirus-encapsulated plasmid to transfect APP695 (K595N, M596L) gene into SH-SY5Y cells and thereby obtain a stable cell line over-expressing Aβ. We used qPCR to monitor the mRNA expression of β-Amyloid Precursor Protein (APP) gene, β-Site APP Cleaving Enzyme 1 (BACE1) gene, we also used Western Blot to detect the APP protein, BACE1 protein, and Aβ1-42, respectively, in addition, the expression of Aβ1-42 in SH-SY5Y cell lysate and cell culture supernatant was measured by Enzyme-Linked Immunosorbent Assay (ELISA). The findings demonstrated APP gene over-expression following APP695 (K595N, M596L) gene transfection, and also indicated the successful establishment of the Swedish mutant cell model.
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ACKNOWLEDGMENTS
The authors thank the Analytical & Testing Center of Shenzhen Institute of Advanced Technology, CAS.
Funding
This research was supported by the National Natural Science Foundation of China (62074155, 62175252), the Basic and Applied Basic Research Foundation of Guangdong Province (2020A1515110142), the Shenzhen Overseas Innovation Team Project (no. KQTD20180413181834876), the Shenzhen Science and Technology Innovation Commission (KCXFZ202002011008124, JCYJ20210324101405016), and the SIAT Innovation Program for Excellent Young Researchers (E25402), the Natural Science Foundation of Ningxia (2020AAC03151), the Innovation and Entrepreneurship Training Program for College Students of Ningxia in 2020 (S202010752048).
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Conceptualization: J. D., Y. Z. and H. Y.; Methodology: H. Z., R. H., J. D.; Formal analysis and investigation: H. Z., Z. Y., R. H., S. H.; Writing—original draft preparation: H. Z., J. R., S. W., H. S.; Writing—review and editing: J. D., Y. Z., H. Y., Y. H.; Funding acquisition: H. Y., P. Z.; Supervision: J. D., Y. Z. and H. Y.
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Huitao Zhang, Hao, R., Yu, Z. et al. Generation of Stable Cell Lines of APP695 (K595N, M596L) Gene Over-Expression via Lentiviral Over-Expression System. Biol Bull Russ Acad Sci 51, 30–36 (2024). https://doi.org/10.1134/S1062359023602586
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DOI: https://doi.org/10.1134/S1062359023602586