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

A technology of homologous recombination and vector, applied in the field of genome editing

Active Publication Date: 2021-06-01
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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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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  • A homologous recombination vector expressing egfp, recombinant cell and its preparation method and application
  • A homologous recombination vector expressing egfp, recombinant cell and its preparation method and application
  • A homologous recombination vector expressing egfp, recombinant cell and its preparation method and application

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preparation example Construction

[0031] The invention provides a method for preparing the homologous recombination vector, comprising the following steps: 1) inserting the left homology arm between the EcoRI restriction site and the HindIII restriction site on the backbone vector Pd-N1 to obtain the vector pd -5'arm; 2) Insert the CMV promoter that starts the eGFP gene into the vector pd-5'arm between the HindIII restriction site and the NheI restriction site to obtain the vector pd-5'arm-CMV; 3) Insert the eGFP gene expression cassette into the vector pd-5`arm-CMV between the NheII restriction site and the BamHI restriction site to obtain the vector pdEgf-G418-5`arm; 4) insert the right homology arm into the vector pdEgfP - The NotI restriction site and the Sal I restriction site on the G418-5`arm were used to obtain the homologous recombination vector pdEgfP-G418-5`arm-3`arm.

[0032] In the present invention, the left homology arm is inserted between the EcoRI restriction site and the HindIII restriction s...

Embodiment 1

[0042] 1. African green monkey embryo kidney epithelial cells were passaged and cryopreserved. African green monkey embryo kidney epithelial cells were obtained from the State Key Laboratory of Livestock Disease Pathogen Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0043] For details on cell culture, subculture and cryopreservation, please refer to "Animal Cell Culture-Basic Techniques and Special Applications" (written by R.I.Freshney, translated by Zhang Jingbo, etc., 2014, sixth edition, Science Press).

[0044] 2. A method for constructing a homologous recombination plasmid expressing eGFP, comprising the following steps:

[0045] (1) Using the African green monkey genome as a template, PCR amplification obtains a 665bp left homology arm, and uses restriction endonucleases EcoR I and Hind III to insert the left homology arm into the vector backbone Pd-N1 to obtain the vector pd -5`arm;

[0046] The double enzyme digestion sy...

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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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/10C12N15/90C12N9/22
CPCC07K14/43595C12N9/22C12N15/66C12N15/85C12N15/907C12N2800/107C12N2800/60
Inventor 常艳燕常惠芸邵军军李扬帆张永光
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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