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Construction method and use of enterovirus 71 type infection model based on hSCARB2 gene knock-in

A gene knock-in and enterovirus technology, applied in the biological field, can solve the problems of transgenic instability, inconsistent copy number of first-built mice, and unstable phenotype of transgenic animal models.

Active Publication Date: 2017-04-05
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transgene by prokaryotic injection is often randomly inserted into the host genome. First, it will lead to inconsistent copy numbers among the first-built mice, and second, it will lead to inconsistent insertion sites.
Therefore, the transgenic animal model will appear unstable in phenotype and unstable in transgene

Method used

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  • Construction method and use of enterovirus 71 type infection model based on hSCARB2 gene knock-in
  • Construction method and use of enterovirus 71 type infection model based on hSCARB2 gene knock-in
  • Construction method and use of enterovirus 71 type infection model based on hSCARB2 gene knock-in

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Embodiment approach

[0081] The present invention also specifically relates to the following embodiments:

[0082] In one embodiment of the present invention, the present invention relates to the method for constructing R26-hSCARB2 mouse, and it mainly comprises the following steps:

[0083] 1) Using embryonic stem cells to knock in the hSCARB2 gene

[0084] Targeting vectors capable of homologous recombination with gene sites in the genome (mutated target genes, homology arms, resistance genes, etc.) were obtained through molecular carrier construction technology; targeting vectors were introduced into embryonic stem cells by electroporation technology, and challenged The drug-resistant embryonic stem cells were taken, and 4 correctly targeted embryonic stem cell clones were screened out by PCR (see Table 1 for primers) and Southern blotting.

[0085] 2) Microinjection of embryonic stem cells into blastocysts and germline inheritance

[0086] Two of the positive embryonic stem cell clones were ...

Embodiment 1

[0117] Example 1: Construction of hSCARB2 targeting vector

[0118] Rosa26 gene can encode a non-essential nuclear RNA in almost all tissues, and can make the inserted exogenous gene express systemically without affecting the expression of other genes. We inserted the hSCARB2 gene cDNA (SEQ ID NO: 1) into the engineered Ai3 targeting vector (Addgene, Madisen L, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13, 133-140 (2010)). The Ai3 vector in turn contains a strong CAG promoter, a termination signal flanked by loxP, and a WPRE element that enhances mRNA transcriptional stability. And insert tdTomato reporter gene (SEQ ID NO:11) at the back of hSACARB2, connect with hSCARB2 through 2A short peptide (coded by SEQ ID NO:10) ( figure 1 ). Under the action of Cre recombinase, the transcription termination signal with loxP is cut off, hSCARB2 and tdTomato can be expressed simultaneously, and the 22nd amino ...

Embodiment 2

[0119] Example 2: Construction of hSCARB2 conditional knock-in mice

[0120] The targeting vector obtained in Example 1 was introduced into C57BL / 6ES cells by electroporation, and the clones selected by G418 were detected by PCR and Southern hybridization to detect 4 correct hSCARB2 gene insertion positive clones ( image 3 ). In order to obtain hSCARB2 knock-in mice, we selected one of the positive clones for blastocyst injection. Using BALB / c blastocysts as recipients, 98 blastocysts were injected and transplanted into ICR pseudopregnant mother mice to obtain 8 chimeric mice. Then the chimeric mice were mated with C57BL / 6 mice, and 10 C57BL / 6 background heterozygous gene knock-in mice were obtained completely derived from targeted ES cells through PCR identification. Furthermore, the heterozygous mice were mated to obtain homozygous gene knock-in mice, named B6-Gt(Rosa)26Sortm1(CAG-hSCARB2-tdTomato), abbreviated as R26-STOP-hSCARB2 mice.

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Abstract

The invention relates to a construction method of an enterovirus 71 type infection model based on hSCARB2 gene knock-in and use of the infection model, and especially provides an effective means for in vivo evaluation of EV71 vaccines and antiviral drugs.

Description

[0001] Technical field: [0002] The invention relates to a construction method and application of an enterovirus EV71 infection model, belonging to the field of biotechnology. The animal model constructed by the invention can be used for the research on the mechanism of EV71 virus infection, the in vivo verification of the protection of EV71 virus vaccine, the research and development of new EV71 vaccine and the screening of antiviral drugs. Background technique [0003] Human enterovirus 71 (EV71) is one of the main pathogens of hand, foot and mouth disease (HFMD), mainly infecting children under 5 years old, and can cause nervous system infection leading to aseptic meningitis , brainstem encephalitis, myocarditis, pulmonary edema, etc. EV71 has caused sporadic outbreaks worldwide, especially in the Asia-Pacific Rim, including Japan, Malaysia, Singapore, inland China, and Taiwan, causing serious social harm to these countries and regions. In my country, since the outbreak ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027A61K49/00
Inventor 范昌发周舒雅刘强吴星陈盼吴曦王佑春毛群颖刘甦苏左琴黄维金李保文梁争论李文辉贺争鸣
Owner NAT INST FOR FOOD & DRUG CONTROL
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