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Preparation method of rrbp1 gene knock-out xenopus tropicalis model, and application

A gene knockout, clawed frog technology, applied in the field of genetic engineering, can solve problems such as no related reports

Active Publication Date: 2020-09-29
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of the rrbp1 gene in Xenopus tropicalis and the corresponding gene knockout strains have not been reported yet.

Method used

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  • Preparation method of rrbp1 gene knock-out xenopus tropicalis model, and application
  • Preparation method of rrbp1 gene knock-out xenopus tropicalis model, and application
  • Preparation method of rrbp1 gene knock-out xenopus tropicalis model, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 CRISPR / Cas9 Gene Knockout Target Site Design

[0036] The genomic DNA sequence and functional domains of the rrbp1 gene of the tropical clawed frog were queried on NCBI. According to the gene editing principle of the CRISPR / Cas9 system, 10 sgRNAs were designed for the first exon (SEQ ID NO.1) of the rrbp1 gene (Table 1). For the design site of sgRNA, see figure 1 .

[0037] Table 1. Ten sgRNA sequences targeting the first exon of rrbp1

[0038]

[0039] In the DNA sequence of the above sgRNA, the sequence in bold is the PAM sequence of the target site recognized by CRISPR / Cas9.

Embodiment 2

[0040] Example 2 Construction and in vitro transcription of sgRNA expression vector

[0041] (1) The sgRNA designed in Example 1 was cloned into the pUC57-Simple-gRNA backbone vector (Addgene, #51306) to construct an sgRNA expression vector. Because the pUC57-Simple-gRNA backbone vector needs to be digested with BbsI, it is necessary to artificially add the sticky end of the BbsI restriction site to the sgRNA and its complementary sequence (add TAGG to the 5' end of the sense strand; add TAGG to the 5' end of the antisense strand) AAAC) to facilitate cloning of sgRNA into the pUC57-Simple-gRNA backbone vector. Anneal the designed sgRNA sequence with the sticky end of the BbsI restriction site and its complementary sequence as a synthetic primer to form a double-stranded DNA fragment with sticky end, and connect it to the pUC57-Simple-gRNA backbone vector digested with BbsI , and then construct the corresponding sgRNA expression vector.

[0042] Table 2 Annealing reaction sys...

Embodiment 3

[0062] The microinjection of embodiment 3 Xenopus tropicalis embryo

[0063] Open the nitrogen gas valve when the tropical clawed frog lays eggs, adjust the pressure and time, and prepare various reagents that need to be injected as needed, inject 300pg / egg according to the Cas9 protein, and inject 100pg of the sgRNA (T1-T10) obtained in Example 2 The amount per egg was injected. According to the needs, select fertilized eggs whose embryo injection period is 1-cell stage. Transfer the embryos to be injected to 0.1×MBS embryo culture medium (composed of 5×MBS (configuration method: weigh NaCl 25.9g, Hepes 11.9g, NaHCO 3 1.0g, KCl 0.4g, MgSO 4 0.5g, Ca(NO 3 ) 2 0.8g, CaCl 2 0.2g, add 800mL deionized water and mix well, adjust the pH to 7.4, set the volume of the volumetric flask to 1000mL, store it at 4°C after autoclaving and dilute it for later use), then transfer it to a glass slide with a straw, according to the injection The need to regulate the position of the em...

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Abstract

The invention relates to a preparation method of a rrbp1 gene knock-out xenopus tropicalis model, and an application. The preparation method comprises the steps of selecting a rrbp1 gene knock-out target point, designing sgRNA, synthetizing a double-stranded DNA fragment, constructing a sgRNA expression vector, performing in vitro transcription to obtain the sgRNA, mixing the sgRNA with Cas9 proteins, performing microinjection into fertilized eggs, and performing screening so as to obtain the rrbp1 gene knock-out xenopus tropicalis model. According to the preparation method disclosed by the invention, a reliable genetic modification animal model is provided for clarifying the functions of a rrbp1 gene in xenopus tropicalis, and a powerful research tool is provided for further studying tumor and cardiovascular diseases related to the rrbp1 gene based on the model; and besides, the method disclosed by the invention provides technical support for innovation of xenopus tropicalis mode animal germplasm resources.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a preparation method and application of a rrbp1 gene knockout tropical clawed frog model. Background technique [0002] Ribosome-binding protein 1 (RRBP1) is one of the important structural proteins of the endoplasmic reticulum (ER), which contains several functional domains and is mainly located on the ER membrane. RRBP1 is essential for the transport and secretion of nascent proteins and ribosome binding in mammalian cells, and it promotes ribosome binding on the ER mainly by enhancing a specific mechanism for collagen biosynthesis at sites of entry into the secretory region. Studies have shown that RRBP1 is involved in the occurrence and development of various diseases or dysfunctions, including cancer and heart-related diseases, which is very important for elucidating the molecular mechanisms of related diseases and developing effective targeted intervention thera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N15/89C12N15/12A01K67/027
CPCC12N15/113C12N15/902C12N15/89C07K14/463A01K67/0276C12N2310/20A01K2217/075A01K2217/15A01K2227/50A01K2267/0375A01K2267/0331
Inventor 齐绪峰张舟蔡冬青刘光辉赵晖郑莉朴圭祥冯珊珊
Owner JINAN UNIVERSITY
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