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Cell strain as well as preparation method and application thereof

A cell line and site technology, applied in botany equipment and methods, biochemical equipment and methods, animal cells, etc., can solve the problems of exogenous gene loss, complicated process, low success rate, etc., and achieve strong fluorescence level, The effect of short time and high integration efficiency

Pending Publication Date: 2021-06-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional method integrates the luciferase expression gene into a vector with a specific promoter element, then transfects it into cells, and obtains a stable cell line through screening. This method easily leads to the loss of foreign genes in cells, and the success rate is low. , and because of the needs of the signaling pathway, other plasmids must be transfected again, the process is complex and time-consuming

Method used

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  • Cell strain as well as preparation method and application thereof
  • Cell strain as well as preparation method and application thereof
  • Cell strain as well as preparation method and application thereof

Examples

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preparation example Construction

[0040] The present invention also provides a preparation method of the cell line, which is to insert the NFAT element and the reporter gene regulated by it into the safe harbor site of the host cell genome by using the Talen or CRISPR / cas9 gene editing method.

[0041] Specifically, the preparation method includes the following steps: co-transfect the host cell with the targeting plasmid and the reporter gene plasmid, and screen the cell line stably expressing the reporter gene after the cultivation is completed to obtain the cell line; To the sequence of the safe harbor site, the reporter gene plasmid contains a reporter gene regulated by the NFAT element and contains a sequence that can be recognized by the targeting plasmid.

[0042] In the embodiment using CRISPR / cas9, the preparation method specifically includes the following steps:

[0043] 1) Construct a targeting plasmid carrying the Cas9 protein;

[0044] 2) constructing a reporter gene plasmid, which includes a NFAT...

Embodiment 1

[0074] Example 1: Design of gRNA targeting AAVS1 site, construction of gRNA-pX458 plasmid and detection of editing efficiency

[0075] Through literature search, the commonly used gRNA targeting sequence at the AAVS1 site was confirmed, and the gRNA forward primer and reverse primer were designed as shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0076] Phosphorylation and pairing of primers: Phosphorylation of primers by T4 PNK, after gradient annealing and pairing, diluted 200 times, used as ligation templates.

[0077] Preparation of linearized vector pX458: Add restriction endonuclease Bbs I to a test tube containing purified vector plasmid pX458, place in a 37°C water bath, digest for 2 hours, and perform 1% agarose gel electrophoresis on the digested product. Gel recovery linear vector. .

[0078] Construction of gRNA-pX458 plasmid: Add 1 μL of diluted paired primers to 50 ng of linearized pX458 vector, then add T4 ligase, and incubate at 25 degrees for 1 hour. ...

Embodiment 2

[0081] Example 2: Construction of the donor plasmid pUC19-NFAT-RE-Luciferase-Puro targeting the AAVS1 site

[0082] In this example, the pUC19-HAL / R plasmid containing the upstream and downstream homology arms of the AAVS1 site was first constructed as an intermediate transition vector, and inserted into Express Luc, Puro and their regulatory sequences, and construct pUC19-NFAT-RE-Luciferase-Puro.

[0083] Linearize pUC19 vector: Add restriction enzymes EcoR I and Hind III to the test tube containing the purified vector plasmid pUC19, place in a 37-degree water bath, digest for 2 hours, and perform 1% agarose gel electrophoresis on the digested product , gel recovery linear vector.

[0084] Retrieving upstream and downstream homology arm sequences: using HEK 293T cell genome as a template, PCR amplified the upstream homology arm (HAL) and downstream homology arm sequence (HAR) of the AAVS1 site. The forward primer and reverse primer for the HAL amplification of the homology ...

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Abstract

The invention relates to the technical field of biology, in particular to a cell strain as well as a preparation method and application thereof, a safe port site of a cell strain genome contains an NFAT element and a reporter gene regulated by the NFAT element, and the NFAT element and the reporter gene regulated by the NFAT element are NFAT-RE-Luciferase genes. The NFAT-RE-Luciferase gene in the cell strain is integrated in the genome in a specific site, and the existing cell strain is mainly obtained through a random integration technology, so that the cell strain has remarkable genome stability, and has higher transcriptional activity and higher fluorescence level compared with the existing random integration cell line, thereby the detection stability and sensitivity of the cell strain are improved. The cell strain provided by the invention also provides a good platform and solution for biological research or drug activity detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell line and its preparation method and application. Background technique [0002] Biotechnology drugs usually have the characteristics of large molecular weight and complex spatial structure, and are highly sensitive to environmental conditions. Due to their complexity, the methods and quality control of biopharmaceuticals are very different from traditional chemical drugs. In terms of consistency, stability, There are certain difficulties in terms of security. Under the control of appropriate regulatory elements, the luciferase reporter gene system uses the activation or inactivation of signal transduction to characterize the changes in cell activity induced by biopharmaceuticals, which has the characteristics of high stability, high accuracy, simplicity and speed. Therefore, the luciferase reporter gene system has become an important alternative to bioassays in the quality con...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/90C12N15/12C12Q1/02C12R1/91
CPCC12N5/0686C12N15/85C12N15/907C07K14/4702G01N33/5041C12N2510/00C12N2800/107
Inventor 路慧丽高阳
Owner SHANGHAI JIAO TONG UNIV
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