A method for in vivo screening of islet beta cell function-promoting drugs

A β-cell and islet technology, applied in the field of diabetes drug screening and genetic engineering, can solve the problems of animal models lacking islet β-cell function, etc.

Active Publication Date: 2021-09-14
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, research on new drug screening for promoting islet β-cell function is still blank, the main reason is the lack of an animal model that can evaluate islet β-cell function conveniently, feasiblely and even automatically in vivo

Method used

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  • A method for in vivo screening of islet beta cell function-promoting drugs
  • A method for in vivo screening of islet beta cell function-promoting drugs
  • A method for in vivo screening of islet beta cell function-promoting drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: Construction of zebrafish model

[0062] 1. Construction of PNPC2-ins-Rcamp1.07 plasmid

[0063] 1.1 Acquisition of the complete gene sequence of zebrafish insulin BAC_ins

[0064] Zebrafish BAC_CH211_69I14 (BACPACResourcesCenter), including the entire sequence of the zebrafish preproinsulin (ins) gene and its immediate upstream and downstream genes (such as figure 1 ).

[0065] 1.2 Zebrafish insulin BAC_ins gene modification

[0066] Use the RecE / RecT homologous recombination system to modify the zebrafish insulin BAC_ins gene. The RecE / RecT homologous recombination system from λ phage is used. RecE is a 5'-3' exonuclease that exposes the single-stranded DNA 3' sticky end; RecT is a single-strand binding protein that can bind to the 3' sticky end of single-stranded DNA and mediate its homologous recombination with homologous sequences on another double-stranded DNA or on the genome , so that the target gene is inserted and replaced (such as figure 2...

Embodiment 2

[0083] Example 2: Identification of transgenic zebrafish models

[0084] In order to further verify that the pancreatic β cells in the Tg(ins:Rcamp1.07) transgenic zebrafish constructed in Example 1 were specifically labeled with Rcamp1.07, the zebrafish whole embryo immunofluorescence staining technique was used to fluorescently label 3 with an insulin antibody. Pancreatic β cells in the Tg(ins:Rcamp1.07) transgenic fish of Tianyu age, to observe whether the signal of Rcamp1.07 is accurately localized in the pancreatic β cells, the results show that the red fluorescent signal of Rcamp1.07 is localized in insulin Antibody-labeled islet β cells (such as Figure 5 ), the Tg(ins:Rcamp1.07) transgenic zebrafish constructed in the present invention is a transgenic zebrafish line β-cell-specific reporterfish that accurately reports the characteristics of pancreatic β cells.

Embodiment 3

[0085] Embodiment 3: the application of transgenic zebrafish model

[0086] 1. Application of Transgenic Zebrafish Islet β Cell Population

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Abstract

The invention discloses a zebrafish model for in vivo screening of islet β-cell function-promoting drugs. The zebrafish model for in vivo screening of pancreatic islet β-cell function-promoting drugs has the coding sequence of the ins gene on the gene BAC_CH211_69I14 replaced with a fluorescent protein sequence , and the fluorescent signals were specifically expressed in islet β cells, which could indicate changes in the calcium ion signal of islet β cells in vivo. The present invention creates the first animal model that can indicate the function of pancreatic islet beta cells in vivo. The transgenic zebrafish line of the present invention can observe the changes of calcium signals in islet beta cells in real time in vivo, and intuitively and accurately report islet beta cells Functional status. Using this transgenic zebrafish line, the functional characteristics of islet β cells can be analyzed conveniently, automatically and with high throughput at the in vivo level, so as to achieve high-throughput screening of drugs that promote islet β cell function, which is of great practical significance and wide range. application prospects.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, to the technical field of drug screening for treating diabetes, and in particular to a method for in vivo screening of drugs for promoting the function of pancreatic beta cells. Background technique [0002] Diabetes is a chronic metabolic disease characterized by hyperglycemia. The main cause of the disease is that the body cannot secrete enough insulin or insulin resistance occurs in the downstream target organ tissue of insulin, which can not effectively lower blood sugar, resulting in elevated blood sugar, diabetes and further serious complications. Research data from around the world shows that in 2013, the number of diabetic patients in the world was estimated to reach 382 million, and the number of diabetic patients in my country reached about 113.9 million, making it the largest country with diabetes. Diabetes has become a global problem that endangers human health, society a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0278A01K2227/10A01K2267/03C12N15/8509
Inventor 刘彦梅赵佳陈良怡
Owner PEKING UNIV
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