Method for obtaining immortalized cell strain through transfection by lentiviral vector mediated SV40 T antigen

Abstract

The invention discloses a method for obtaining an immortalized cell strain through transfection by a lentiviral vector mediated SV40 T antigen, and belongs to the field of cytobiology. Concretely, anSV40 T lentiviral expression vector capable of realizing efficient and fast transfection is built; through engineering cell packaging on the vector, lentiviral particles expressing the SV40 T antigencan be obtained and are used for transfecting primary generation separation cells; and further, the immortalized cell strain is obtained through the zeocin resistance characteristic screening. The method for building the immortalized cell strain disclosed by the invention has the characteristics of high efficiency, convenience and high speed; the price is low; and the method is suitable for beingpopularized and used.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a method for obtaining immortalized cell lines through lentiviral vector-mediated SV40 T antigen transfection. Background technique [0002] In cell biology research, most primary isolated cells, as terminally differentiated cells, have a limited time of culture and passage in vitro, and after a certain number of passages, they will enter the senescent stage and cannot continue to proliferate. However, repeated isolation of primary cells is not only time-consuming and laborious, but also there are differences among batches of cells, which cannot maintain the stability of cell physiological indicators. [0003] The inactivation of some tumor suppressor genes P53 and pRB and the activation of telomerase are necessary conditions for the immortalization of human cells. Mammalian cells use several methods to induce cell immortalization (Cell immortalizing), including the use o...

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Problems solved by technology

[0004] At present, some methods have been used to mediate the transfection of the SV40 large T antigen gene, such as transfecting the target primary cells with the eukaryotic expression plasmid vector of the SV40 large T antigen gene through liposome mediation and screening to obtain immortalization Cell line method, but its transfection efficiency is low, screening is difficult, and some difficult-to-transfect cells are difficult to obtain stable expression and establish cell lines

Other constructed adenovirus or lentivirus SV40 T expression vectors lack convenient screening methods after transfection, such as incubator culture screening with special temperature control

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