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.
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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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