A homologous recombination vector expressing egfp, recombinant cell and its preparation method and application

Abstract

The invention provides a homologous recombination vector expressing eGFP, which belongs to the field of genome editing technology. The homologous recombination vector expressing eGFP includes a vector backbone and an insert segment, and the insert segment includes a left homology arm, a CMV promoter, An eGFP expression frame and a right homology arm; the nucleotide sequence of the left homology arm is shown in SEQ ID No.1; the nucleotide sequence of the right homology arm is shown in SEQ ID No.2. After the homologous recombination vector described in the present invention and the CRISPR / Cas9 targeting vector are co-transfected into vero cells, the vero cells are screened by G418 to obtain vero cells that integrate eGFP at a specific point and stably express eGFP. The cell construction method described in the present invention supports site-specific integration and stable expression of foreign genes.

Description

technical field [0001] The invention belongs to the technical field of genome editing, and in particular relates to a homologous recombination vector expressing eGFP, a recombinant cell and a preparation method and application thereof. Background technique [0002] CRISPR-Cas9 (Clustered Regularly Interspersed Short Palindromic Repeats-Cas9, CRISPR-Cas9) uses target-specific RNA to bring the Cas9 endonuclease to a specific target in the genome to achieve modification (deletion or insertion) of specific genetic sites , the technology has been rapidly developed in the field of life sciences. CRISPR-Cas9 is a method that can be used for site-specific gene targeting after zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Compared with ZFNs and TALENs, this method has the following advantages: (1) The structure is simple, and only a single guide RNA (single guide RNA, sgRNA) is needed to complete the target recognition; (2) The targeting ...

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Problems solved by technology

[0003] Engineering cell lines have been greatly developed in recent years. Although more efforts are devoted to increasing the expression of recombinant proteins, how to up-regulate, down-regulate, knock out or stably express Certain cellular endogenous or exogenous genes that improve cell growth characteristics, leading to increased cell proliferation and maximum viable cell density, and high-yield expression of exogenous proteins or viruses that maximize yield and reduce associated costs are still considered Many scientists and vaccine manufacturers are concerned

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