Vector capable of fast acquiring bastard halibut stable-transfected cell line

Abstract

The invention provides a vector capable of achieving efficient and stable transfection in bastard halibuts. The vector adopts a Tol2 transposon, and the Tol2 transposon combines with the plasmid pCS-TP for synthesizing transposase to perform transfection can evidently increase the exogenous gene integrating ability of bastard halibut cells. A neomycin resistance screening marker is added to the vector, so that many recombinant cells can be obtained only by a traditional method combining liposome transfection and resistance screening, and the screening efficiency of the bastard halibut cells being screened to the stable-transfected cell line is increased greatly. An EGFP fluorescent label is added to the vector, so that the expression condition of exogenous genes transfected into the cell line can be conveniently showed in a tracing manner. More importantly, the vector uses an optimized bastard halibut beta-actin promoter to replace a CMV promoter commonly used in a traditional mammal expression vector, the vector more suitable for bastard halibut cell line exogenous gene expression is obtained creatively, and accordingly the exogenous genes can be efficiently and stably expressed in various bastard halibut cell lines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a carrier capable of rapidly obtaining a stable transfected cell line of flounder. Background technique [0002] Cell transfection is a specialized technology for introducing exogenous molecules such as RNA and DNA into cells, and has become a routine method for studying gene functions in molecular biology and cell biology. At present, the more commonly used methods for cell transfection include liposome method, electroporation method and virus infection method. The electroporation method relies on instantaneous high-pulse voltage to break down the cell membrane, thereby introducing foreign plasmids into the cells. It has a wide range of applications, but the operation is relatively cumbersome, and the lethality of cells after transfection is high. The cells required for transfection And the amount of DNA is large, so it is rarely applicable in the process of cell line t...

AI Technical Summary

Problems solved by technology

However, the following problems are still encountered in the process of using liposome method to transfect flounder cells: ① It is difficult to integrate the exogenous gene into the cell genome DNA after the foreign gene is transferred into the flounder cell line, so it is difficult to rely on the traditional Steadily transfected flounder cell lines obtained by screening after liposome transfection

②Exogenous genes successfully transfected into flounder cells are difficult to maintain high-efficiency expression, and the expression efficiency of foreign genes in most successfully transfected flounder cells will disappear within a week, which is significantly lower than that of mammalian cells Expression efficiency of exogenous gene after transfection

[0004] The main reasons for the analysis are as follows: ①After the recombinant plasmid is transferred into the flounder cells, with the prolongation of the screening time, most of the recombinant plasmids will be gradually lost with cell division, and a small part of the remaining plasmids is difficult to integrate into the flounder cells correctly Genomically, this makes it difficult to screen for stable recombinant cell lines

②The CMV promoter on the commonly used mammalian expression vector cannot efficiently and persistently promote the expression of downstream genes in flounder cells. Although the initial efficiency is high, its activation efficiency gradually decreases with time

[0005] Restricted by the above reasons, for a long time, liposome transient transfection has been used in the process of using flounder cell lines to study its gene function, but the stable transfection method commonly used in mammalian cell lines cannot be used. , so it is impossible to obtain a flounder cell line stably transfected with foreign genes, which greatly limits the functional research of gene regulation in flounder

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