Preparation method of liver cells with low expression or no expression of PERV

A liver cell, low-expression technology, applied in the field of transgenic technology, can solve problems such as reducing the risk of PERV infection, and achieve the effect of saving preparation time and improving preparation efficiency

Inactive Publication Date: 2015-12-16
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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Problems solved by technology

The existing PERV inactivation or elimination methods have failed to reduce the risk of PERV infection

Method used

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  • Preparation method of liver cells with low expression or no expression of PERV
  • Preparation method of liver cells with low expression or no expression of PERV
  • Preparation method of liver cells with low expression or no expression of PERV

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Embodiment Construction

[0031] The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

[0032] The preparation method of the hepatocytes with low expression or no expression of PERV of the present invention comprises the following steps:

[0033] 1. Design and preparation of recombinant vectors

[0034] (1) Preparation of recombinant vectors for RNA interference

[0035] RNA interference (RNAi) refers to the highly conserved phenomenon in the evolution process, which is induced by double-stranded RNA (double-stranded RNA, dsRNA), and the phenomenon of efficient and specific degradation of homologous mRNA.

[0036] When exogenous genes such as viral genes, artificially transferred genes, and transposons are randomly integrated into the host cell genome and transcribed by the host cell, some dsRNA is often produced. The host cell immediately responds to these dsRNAs, and the endonuclease Dicer in the cytoplasm cuts the d...

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Abstract

The invention discloses a preparation method of liver cells with low expression or no expression of PERV. The preparation method includes the following steps: (1), respectively preparing a recombinant vector containing siRNA applied to RNA interference technology, a recombinant vector containing gRNA applied to CRIPRS/Cas9 technology and a vector containing Cas9 applied to CRIPRS/Cas9 technology; (2), jointly transfecting the three vectors to porcine somatic cells, culturing the transfected porcine somatic cells, and screening and identifying to obtain monoclonal genetically-modified porcine somatic cells; (3), utilizing somatic cell nuclear transfer technology to obtain genetically-modified pig containing nuclear genes of the genetically-modified porcine somatic cells; (4), extracting liver cells of the genetically-modified pig. According to the preparation method, knockout before transcription and inhibition after transcription of PERV genes are realized, and the technical scheme is effective and feasible.

Description

technical field [0001] The invention relates to a transgenic technology, in particular to a method for preparing hepatocytes with low expression or no expression of PERV. Background technique [0002] Today, severe hepatitis, liver failure and liver cancer are the main causes of death from liver diseases in my country, and liver transplantation is the most effective method for treating such diseases. However, there is already a growing shortage of human homogeneous organ transplant donors, and most patients lose their lives while waiting for organ donors. Finding alternative means for patients with liver failure before liver transplantation has become a top priority. Bioartificial liver (BAL) is currently recognized at home and abroad as an artificial liver support system that is closest to the natural liver and has the most comprehensive functions. The bioartificial liver consists of reactors and cell materials, and hepatocytes are the core material of the entire bioartif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A01K67/027
Inventor 张健民高毅
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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