Zebra fish model for screening medicine used for promoting function of pancreatic beta cell in vivo

A zebrafish and β-cell technology, applied in the field of diabetes drug screening and genetic engineering, can solve the problems of animal models lacking islet β-cell function, and achieve the effect of promoting high-throughput screening and having wide practical significance.

Active Publication Date: 2017-05-31
PEKING UNIV
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  • 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 th

Method used

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  • Zebra fish model for screening medicine used for promoting function of pancreatic beta cell in vivo
  • Zebra fish model for screening medicine used for promoting function of pancreatic beta cell in vivo
  • Zebra fish model for screening medicine used for promoting function of pancreatic beta cell in vivo

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 Obtaining the complete gene sequence of zebrafish insulin BAC_ins

[0064] Zebrafish BAC_CH211_69I14 (BACPAC Resources Center), 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 reporter fish 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 zebra fish model for screening a medicine used for promoting the function of a pancreatic beta cell in vivo. The coded sequence of an ins gene on a gene BAC_CH211_69I14 of the zebra fish model is replaced by a fluorescent protein sequence, and a fluorescence signal is specifically expressed in the pancreatic beta cell to indicate the change of the calcium ion signal in the pancreatic beta cell in vivo. The invention creates the first animal model capable of indicating the function of the pancreatic beta cell in vivo. The change of the calcium signal in the pancreatic beta cell is observed in real time in vivo through a transgenic zebra fish system, and the state of the function of the pancreatic beta cell is intuitively and accurately reported. By using the transgenic zebra fish system, the functional characteristics of the pancreatic beta cell are conveniently automatically analyzed at high throughput on the in-vivo level, so that the zebra fish model has important practical significance and a broad application prospect for the high-throughput screening of the medicine used for promoting the function of the pancreatic beta cell.

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 zebrafish model 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, ...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0278A01K2227/10A01K2267/03C12N15/8509
Inventor 陈良怡
Owner PEKING UNIV
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