An auxiliary plasmid for efficiently constructing human stable expression cell lines and its construction method

An auxiliary plasmid and stable expression technology, applied in the field of genetic engineering, can solve the problem of low expression of exogenous genes, achieve the effect of simple construction process, efficient construction, and increased expression

Active Publication Date: 2020-04-24
重庆高圣生物医药有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is firstly to solve the problem of low exogenous gene expression in the construction of human stable expression cell lines through eukaryotic plasmid transfection, and to provide an auxiliary plasmid for efficient construction of human stable expression cell lines

Method used

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  • An auxiliary plasmid for efficiently constructing human stable expression cell lines and its construction method
  • An auxiliary plasmid for efficiently constructing human stable expression cell lines and its construction method
  • An auxiliary plasmid for efficiently constructing human stable expression cell lines and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0018] Example Helper plasmids to efficiently construct human colon cancer cell lines stably expressing p53 gene

[0019] (1) Construction of helper plasmid

[0020] The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid ID: 42230, hereinafter referred to as pSpCas9(BB)) was digested with BbSI, and after 1 hour in a water bath at 37°C, electrophoresed on 1% agarose to recover the digested product ( TAKARA gel recovery kit).

[0021] The enzyme digestion system is as follows:

[0022]

[0023] Use the online tool ZiFiT Targeter version 4.0 to design oligonucleotides targeting human genome-specific target site sequences, specifically:

[0024] Specific target site sequence: 5'-GGAGGGGAACATCACATACC-3' (SEQ ID NO.1).

[0025] The corresponding oligonucleotide pair is: Oligo(+):5'-caccGGAGGGGAACATCACATACC-3'(SEQ ID NO.2);

[0026] Oligo (-): 5'-aaac GGTATGTGATGTTCCCCCTCC-3' (SEQ ID NO. 3).

[0027] Anneal the two oligonucleotides to form a short double-stranded DNA w...

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Abstract

The invention discloses a helper plasmid for efficiently establishing a human stable expression cell strain and an establishment method thereof. The helper plasmid and an exogenous gene cotransfect a human recipient cell, and the helper plasmid can manually form 167 nicks on a human genome specificity target set sequence to provide 167 integration hotspots for the exogenous gene, so that the efficiency of the integral entrance of the exogenous gene into a human genome is remarkably improved, and further the expression quantity of the exogenous gene is increased. The helper plasmid disclosed by the invention is simple in establishment process and cheap, can be independently established in an ordinary laboratory, can directly cotransfect the human recipient cell with different exogenous genes, is simple in operation method, can remarkably improve the efficiency of establishing the human stable expression cell strain, can realize the efficient establishment of various human cell models, and facilitates the study of human disease pathogenesis and the development of gene therapy.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an auxiliary plasmid for efficiently constructing a human stable expression cell line and a construction method thereof. Background technique [0002] At present, people are not very clear about the pathogenesis of many human diseases, and it is impossible to study the whole process of disease occurrence in vivo. Our experience is not only limited in time and space, but also many experiments are morally and methodically limited, so we need to use cell models and animal models to complete this type of research. Most of the current research is only based on the level of rodent cell lines, and there are significant differences between human cells and rodent cells. There are certain differences between human cells and rodent cells in the two stages of immortalization and cell transformation. Mammalian cells are prone to spontaneous transformation in vivo, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66
Inventor 周勇温平申友锋唐秋月何燕刘兰兰向垚艮陈思源
Owner 重庆高圣生物医药有限责任公司
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