Tetracycline stringent controlled eucaryon expression vector

Abstract

The invention provides a tetracycline strictly controlled eukaryotic expression vector. Following DNA elements including hCMV, rtTA2-M2, IRES, EGFP-Puro, element (Luc) of coding luciferase and Ptetbi-RFC are respectively introduced and are used for the expression and screening of exogenous interest genes in eukaryotic cells. The vector is strictly controlled by Tet or Dox after being transfected. And the expression level of the genes is quantitatively regulated through the amount of the added Tet or Dox. The expression level of the interest genes can be indirectly detected through the detection of the activity of Luc luciferase. The transfection efficiency of the vector can be completed through the detection of the proportion of green fluorescyte. The stably transfected cells are sorted by a flow cytometry. Thus, the expression of the interest genes is ensured to be simple and convenient. And in a stable cell line obtained through screening, the interest genes are integrated with a host genome. The tetracycline strictly controlled eukaryotic expression vector has long stable expression characteristics and advantages.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a eukaryotic expression carrier strictly regulated by tetracycline, in particular to the convenient recombination of exogenous genes of different interests into the carrier, and the screening of positive cells transfected with the carrier. Background technique [0002] The rapid development of functional genomics research requires a tightly regulated gene expression system, so that the expression of foreign genes in host cells can be precisely regulated, so that their functional research can proceed smoothly. In recent years, the gene expression system regulated by tetracycline (Tet) has been widely used. Tet-induced expression systems are divided into two types: one is the Tet-closed type, and the other is the Tet-opened type. The former is constitutively expressed in the absence of Tet and its derivative doxycycline (Dox), and is regulated in the presence of Tet or Dox...

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Problems solved by technology

[0003] Although there have been many reports of Tet or Dox-induced expression systems (Georgievska, B., J.Jakobsson, E.Persson, C.Ericson, D.Kirik and C.Lundberg.2004.Regulated delivery of glial ce11line-derived neurotrophic factor into rat striatum, using atetracycline-dependent lentiviral vector. Hum Gene Ther. 15: 934-944.; Aurisicchio, L., A. De Tomassi, N. La Monica, G. Ciliberto, C. Traboni and F. Palombo, 2005. Regulated and liver-specific tamarin alpha interferon gene delivery by a helper-dependent adenoviral vector.J Virol.79:6772-6780), but the problem is that the replacement of the target gene regulated by Tet or Dox is inconvenient; for example, after high-throughput To obtain a lot of interest genes to be detected by using the method of screening, it is necessary to design a recombination scheme according to the sequences and restriction sites of different genes, and use traditional restriction methods to construct a Tet or Dox regulatory system, which requires a lot of work, time-consuming and labor-intensive

In addition, in the study of gene function, for the transfer, expression and acquisition of stable expression of the gene of interest, corresponding high-efficiency screening markers are required, which play a crucial role in the success of the work; the existing Screening markers have not yet achieved concise, fast, and convenient screening effects

Finally, the currently reported Tet or Dox inducible expression vectors have eukaryotic replicons, which can replicate autonomously after being transferred into eukaryotic cells; although the effect is fast, there is no exogenous gene integrated into the host genome, and the expression is not stable enough , After a long period of time (several months), there will be weakening and loss of vector expression, which will affect the long-term study of gene function

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