RXRalpha (Homo Sapiens Retinoid X Receptor alpha) gene knockout cell system with stable and low expression of RXRalpha protein and preparation method of RXRalpha gene knockout cell system

A gene knockout and α gene technology, applied in the field of biological preparation, can solve the problems of low probability of occurrence and high fidelity, achieve good reproducibility, simplify procedures, improve reliability and research efficiency

Active Publication Date: 2018-11-06
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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AI Technical Summary

Benefits of technology

Inventors have developed an improved technique called Cell Line that allows scientists to create high amounts of specific proteins from small samples taken from different sources without being affected by other factors like environmental conditions or how they are used during experiments. These techniques improve our understanding about various aspects of biology such as regulation mechanisms involved in signal transmission between neurons and developmental processes associated therewith.

Problems solved by technology

Technics Problem addressed by this patents relates to finding reliable ways to create small amounts of these proteins that could potentially treat various types of cancer, such as those caused by certain enzymes called ribosomal degrading factors.

Method used

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  • RXRalpha (Homo Sapiens Retinoid X Receptor alpha) gene knockout cell system with stable and low expression of RXRalpha protein and preparation method of RXRalpha gene knockout cell system
  • RXRalpha (Homo Sapiens Retinoid X Receptor alpha) gene knockout cell system with stable and low expression of RXRalpha protein and preparation method of RXRalpha gene knockout cell system
  • RXRalpha (Homo Sapiens Retinoid X Receptor alpha) gene knockout cell system with stable and low expression of RXRalpha protein and preparation method of RXRalpha gene knockout cell system

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Embodiment 1

[0035] (1) Setting of RXRα gene knockout target site and design of gRNA

[0036] According to the RXRα gene sequence, select the common exon4 of RXRα-a / b / c as the knockout target sequence, and design the CRISPR target site, such as figure 1 The position indicated by the arrow is selected for knockout site;

[0037] The selected knockout target sites are bases 1-180 of exon 4 of the RXRα gene, and the DNA was designed with bases 44-66 (CTTCAAGCGGACGGTGCGCAAGG), 79-101 (CCTGCCGCGACAACAAGGACTGC), and 106-128 (TTGACAAGCGGCAGCGGAACCGG) Oli gos primers to synthesize double-stranded gRNA.

[0038] Use online software to design CRISPR knockout gRNA and synthesize complementary DNAOligos primers, see figure 2 The indicated gRNA sequence design.

[0039] figure 2 Among them, Guid#1-#5 are the high-scoring knockout target sites selected by the software design respectively. In this project, Guid#1, #2, and #5 are selected to design gRNA synthesis primers, and the corresponding prim

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Abstract

The invention belongs to the technical field of biological preparation and in particular relates to an RXRalpha (Homo Sapiens Retinoid X Receptor alpha) gene knockout cell system with stable and low expression of an RXRalpha protein and a preparation method of the RXRalpha gene knockout cell system. The cell system is an RXRalpha gene knockout cell system; the knockout base number is an integral multiple of a number except 3; RXRalpha protein expression in a cell is remarkably reduced, the genotype is stable and can be passed to next generations, the stability of the RXRalpha low-expression cell system is greatly improved, and stable generation passage and continuous culture of the cell system can be facilitated. The invention provides the preparation method of the cell system. The preparation method comprises the following steps: according to RXRalpha gene sequence information, designing a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) knockout gRNA (Guide Ribonucleic Acid) sequence, establishing a gRNA expression vector, and carrying out in-vitro cell level detection on gRNA shearing activity; by using a nuclear transferring method, carrying out cotransfectionon an immortalized cell of human neuroblastoma, that is, an SK-N-SH cell, by using cas9 and the gRNA expression vector, carrying out G418 medicine screening so as to obtain stable cell cloning, carrying out a PCR (Polymerase Chain Reaction) and gene sequencing to identify that the cell is cell cloning of which the RXRalpha gene is knocked out for an integral multiple of a number except 3, therebyobtaining the RXRalpha gene knockout cell system with stable and low expression of the RXRalpha protein.

Description

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Claims

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Application Information

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Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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