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Method for incubating transgenic zebrafishes with intestinal-specific expression of red fluorescence

A technology of red fluorescence and red fluorescent protein, which is applied in the field of transgenic zebrafish cultivation, can solve the problem of reducing the efficiency of screening and breeding of transgenic zebrafish, and achieve the effect of simple and easy control of the implementation process and meeting the requirements of integrity and integrity

Inactive Publication Date: 2020-09-04
ZHEJIANG UNIV
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Problems solved by technology

[0005] During the development of zebrafish embryos, the position of intestinal development is very close to that of yolk, and the intestinal-specific fluorescence of transgenic zebrafish is easily affected by the strong autofluorescence of yolk, thus reducing the need for screening and breeding during the cultivation of transgenic zebrafish. s efficiency

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  • Method for incubating transgenic zebrafishes with intestinal-specific expression of red fluorescence
  • Method for incubating transgenic zebrafishes with intestinal-specific expression of red fluorescence
  • Method for incubating transgenic zebrafishes with intestinal-specific expression of red fluorescence

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Experimental program
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specific Embodiment

[0046] 1. The Pifabp promoter was amplified from the zebrafish intestinal tissue genome

[0047] 1. Zebrafish genome acquisition

[0048] (1) Take one dead adult zebrafish, use surgical instruments to dissect to obtain intestinal tissue, cut it into pieces, put it into a mortar, pour liquid nitrogen into it, and grind;

[0049] (2) Add 1mL PBS, mix well and transfer to 1.5mL EP tube;

[0050] (3) Add 100 μL 10% SDS and 5 mg proteinase K, and incubate at 37° C. for 1 hour;

[0051] (4) After centrifuging at 12000rpm for 1min, transfer the supernatant to a new 1.5mL EP tube, add an equal volume of phenol:chloroform (1:1) and let stand for 5min;

[0052] (5) Centrifuge at 12000rpm for 10min, transfer the supernatant to a new 1.5mL EP tube, add twice the volume of a mixture of 75% ethanol and 3M sodium acetate (9:1) pre-cooled on ice, and place on ice for 5min , to be precipitated;

[0053] (6) Centrifuge at 12000 rpm at 4°C for 10 min, add 1 mL of pre-cooled 75% ethanol to wa...

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Abstract

The invention relates to an incubation method for transgenic zebrafishes, and aims to provide a method for incubating transgenic zebrafishes with intestinal-specific expression of red fluorescence. According to the invention, by observing the expression of embryonic red fluorescent protein gene, zebrafish embryos with intestinal-specific fluorescence are selected, and hybrid offsprings are obtained by artificial feeding. The stably genetic zebrafishes with intestinal expression of red fluorescent protein are obtained by selfing of the hybrid offsprings, and then parent zebrafishes corresponding to embryos with 100% intestinal expression of red fluorescent protein are screened as double integration pure lines for seeding and preservation after test cross with wild type zebrafishes. The method of the present invention is simple and easy-to-control in implementation process, and can obtain a promoter with intestinal-specific expression and zebrafish strains with red fluorescent protein highly expressed in the intestine. The obtained zebrafish strains are used for the study of zebrafish intestinal function and can be observed for expression characteristics of intestinal red fluorescentprotein at any time, thereby meeting the integrity and completeness requirements on study of zebrafish intestinal function.

Description

technical field [0001] The present invention relates to a method for cultivating transgenic zebrafish, in particular to a method for cultivating zebrafish expressing red fluorescent protein in the intestinal tract, and a special promoter for the cultivating method. Background technique [0002] Zebrafish (Denio Rerio) belongs to the family Cyprinidae. It is easy to raise and has strong reproductive ability. The fish eggs are fertilized and developed in vitro, and the fertilized eggs develop rapidly. The primordia of various organs are basically formed during the embryonic period, and the growth cycle is short. Large-scale gene mutation and screening, zebrafish embryos are transparent, and optical instruments can be used to visualize and analyze physiological processes in vivo. These characteristics make it one of the important vertebrate models. [0003] So far, only a few gut-specific promoters have been found in zebrafish, including the promoter of the ifabp gene. The zeb...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65A01K67/027
CPCA01K67/0275A01K2207/05A01K2227/40A01K2267/03A01K2267/0393C12N15/65C12N15/8509C12N2800/106C12N2830/008
Inventor 单颖李肖梁方维焕
Owner ZHEJIANG UNIV
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