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基于遗传逻辑电路的合成生物学,利用耐药性表达,MCF-7癌细胞系中的BCRP泵

Synthetic Biology Based on Genetic Logic Circuit, Using the Expression of Drug Resistance, BCRP Pump in MCF-7 Cancer Cell Line.

作者信息

Gerami Marzieh, Emadi-Baygi Modjtaba, Eshghi Mohammad, Elahian Fatemeh, Heidari Razieh, Hosseinzadeh Mehdi

机构信息

Department of Computer Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran.

Department of Genetics, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran.

出版信息

Iran J Pharm Res. 2020 Summer;19(3):195-205. doi: 10.22037/ijpr.2020.111970.13459.

Abstract

Biological circuits are developed as biological parts within a cell to carry out logical functions resembling those studied in electronics circuits. These circuits can be performed as a method to vary cellular functions, to develop cellular responses to environmental conditions, or to regulate cellular developments. This research explored the possibility of synthetic biology based on the genetic logic circuit and (not ) using the inducible expression of the both BCRP drug resistance pump and its specific shRNA in MCF-7 cancer cell line utilizing the third generation of lentiviral vectors. The accuracy of the output of the proposed circuit for living cells, was confirmed by the results of the Real-Time PCR and flow cytometry at the RNA and protein levels. At the RNA level, the effect of the inducers on the BCRP gene expression and silencing were investigated by real-time PCR. Furthermore, at the protein level, induction of the expression of the BCRP pump resulted in driving out of the substrate from inside the cells leading to the decrease of the fluorescent emission from the transfected cells. We successfully designed and implemented the genetic logic circuit and (not ) using the inducible expression of the both BCRP drug resistance pump and its specific shRNA in MCF-7 cancer cells.

摘要

生物电路是作为细胞内的生物部件而开发的,用于执行类似于电子电路中所研究的逻辑功能。这些电路可以作为一种改变细胞功能、使细胞对环境条件产生反应或调节细胞发育的方法来发挥作用。本研究探索了基于遗传逻辑电路,利用第三代慢病毒载体,在MCF-7癌细胞系中(不)使用BCRP耐药泵及其特异性shRNA的诱导表达进行合成生物学研究的可能性。通过实时PCR和流式细胞术在RNA和蛋白质水平上的结果,证实了所提出的活细胞电路输出的准确性。在RNA水平上,通过实时PCR研究了诱导剂对BCRP基因表达和沉默的影响。此外,在蛋白质水平上,BCRP泵表达的诱导导致底物从细胞内被排出,从而导致转染细胞荧光发射的降低。我们成功地设计并实现了在MCF-7癌细胞中(不)使用BCRP耐药泵及其特异性shRNA诱导表达的遗传逻辑电路。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/23dc/7757972/2d506ca4b167/ijpr-19-195-g001.jpg

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