Construction method and application of transgenic rat specifically expressing hMRGPRX4

A construction method, transgenic technology, applied in the direction of genetic engineering, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the problems of genes without functional homology, and genetic methods cannot be used

Active Publication Date: 2021-01-26
海湃泰克(北京)生物医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, for the development of drugs for the treatment of human cholestatic pruritus, since MRGPRX4 is a primate-specific receptor, there is no functional homologous gene in both mi

Method used

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  • Construction method and application of transgenic rat specifically expressing hMRGPRX4
  • Construction method and application of transgenic rat specifically expressing hMRGPRX4
  • Construction method and application of transgenic rat specifically expressing hMRGPRX4

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Embodiment 1

[0045] The present invention constructs a humanized rat by expressing human MRGPRX4 in rat itch sensory neurons, and provides an animal model for studying the function of MRGPRX4 and screening antipruritic drugs targeting human MRGPRX4. The specific method is as follows:

[0046] (1) Construction of hMRGPRX4-Cre rats

[0047] In the rat MrgA gene, hMRGPRX4-3xFlag-P2A-iCre-WPRE-pA (SEQ ID NO: 6) is used to replace the nucleotide sequence corresponding to the 28th amino acid on the MrgA gene exon 2 (exon2) to the terminator . The sgRNAs were designed in intron 1 (intron1) and exon 2 of the MrgA gene, respectively. Based on the design principle of sgRNA, multiple sgRNAs are designed in the target site area. The sgRNA (5′-TCCGGTTAGGAGTAAATTCC-3′ and 5′-TGAGACAGCGGAAAACACGG-3′) and Cas9 gene were connected to the plasmid vector pCS with T7 promoter and transcribed in vitro to obtain gRNA and Cas9 mRNA for microinjection.

[0048] According to the target gene sequence, the 5' an...

Embodiment 2

[0085] Figure 4 It further proved that the hMRGPRX4-Cre; lsl-hMRGPRX4-mScarlet transgenic rat has more advantages as an animal model. Such as Figure 4 As shown in a, hMRGPRX4-Cre homozygous rats were mated with a reporter rat, and under the action of Cre recombinase, a red fluorescent protein tdTomato would be expressed in the Cre-positive cells of the progeny rats. Extract and cultivate the DRG neurons of the offspring rats, such as Figure 4 As shown in b, the arrow shows the DRG neurons expressing tdTomato, indicating that the Cre recombinase is expressed and functional, and it also theoretically indicates that hMRGPRX4 is expressed; Figure 4 As shown in c, calcium imaging results showed that the addition of DCA did not cause obvious calcium signals in these neurons, indicating that although hMRGPRX4 was expressed, it may not mediate the response caused by DCA in DRG neurons due to the relatively low expression level. This also shows that it is necessary to increase t...

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Abstract

The invention provides a construction method and application of a transgenic rat specifically expressing hMRGPRX4. A humanized rat is successfully constructed by expressing hMRGPRX4 in a rat itching sensation neuron. The specific method comprises the following steps: respectively constructing transgenic rat strains hMRGPRX4-Cre and lsl-hMRGPRX4-mScarlet, and mating the two transgenic rat strains to obtain the transgenic rat specifically expressing hMRGPRX4. The invention provides an animal model for researching the function of hMRGPRX4 and screening anti-pruritus drugs targeting hMRGPRX4.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a transgenic rat specifically expressing hMRGPRX4 and its application. Background technique [0002] Patients with chronic liver disease are often accompanied by unbearable itching, and up to 60% of patients with primary biliary cholangitis (PBC) have symptoms of chronic itching, seriously affecting the quality of life of patients. However, there is still a lack of effective drugs for relieving or treating the pruritus. Recent studies have shown that human MRGPRX4 mediates bile acid-induced cholestatic pruritus. Cholic acid (CA) and its derivatives can activate hMRGPRX4; the content of cholic acid in the serum of chronic liver disease patients with pruritus is significantly higher than that of normal people; cholic acid and hMRGPRX4 agonists can induce obvious itching. Therefore, hMRGPRX4 is a drug target for the treatment of human cholestatic pruritus, a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65A01K67/027
CPCC12N15/8509C12N15/65A01K67/0278C07K14/723A61K49/0008C12N2800/107A01K2267/03A01K2267/0393A01K2227/105A01K2217/072A01K2207/15
Inventor 李毓龙赵天军雷晓光
Owner 海湃泰克(北京)生物医药科技有限公司
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