Method for obtaining holonephros expression green fluorescent protein white zebra fish

Abstract

The invention discloses a method for obtaining a holonephros expression green fluorescent protein zebra fish. The method can overcome the defects that green fluorescent protein of an existing zebra fish line can not be evenly and simultaneously expressed in a whole original glomerulus, a tubule and a catheter. Green fluorescent protein of a zebra fish in the method can be simultaneously expressed in a whole original glomerulus, an original kidney tubule and an original kidney catheter. The method is a crossbreeding method according to which an Na, K-ATPase alpha1A4: GFP line zebra fish and a wt1b: GFP line zebra fish are mutual parents. The method for obtaining the holonephros expression green fluorescent protein zebra fish includes the following steps of hybridization parent selection and breeding, matching and breeding, hybridization character sieving, hybridization filial generation breeding and hybridization character fixing and retaining. The method for obtaining the holonephros expression green fluorescent protein zebra fish is a separation element hybridization stable integration method, the implementation process is easy to control, a zebra fish line of which green fluorescent protein can be simultaneously, evenly and efficiently expressed in a whole original glomerulus, an original kidney tubule and an original kidney catheter is obtained and the requirement for integrality and completeness of study on the original kidney functions of the zebra fish is met.

Description

technical field

[0001] The invention relates to a zebrafish breeding method, in particular to a breeding method for obtaining whole kidneys expressing green fluorescent protein zebrafish, and belongs to the field of biotechnology. Background technique

[0002] In 1962, Shimomura et al. first discovered the bioluminescent protein aequorin in Aequoria victoria, and observed a by-product of light green fluorescence under ultraviolet irradiation - green fluorescent protein (Green fluorescent protein). Fluorescent Protein, GFP). In 1974, Shimomura et al. completed the purification of GFP, and Prasher et al. cloned its gene 18 years later. In 1994, Chalfie et al. realized the expression of GFP in E. coli cells and Caenorhabditis elegans. As an important biomarker protein, GFP can self-catalyze to form a chromophoric structure and emit green fluorescence under ultraviolet or blue light excitation. Because GFP has many advantages such as stable fluorescence, safety and non-toxicit...

Examples

Embodiment 1

[0025] Such as Figure 1 ~ Figure 4 As shown, the whole kidney expressing green fluorescent protein zebrafish was obtained by the method of the present invention.

[0026] 1. Breeding of hybrid parents

[0027] Na,K-ATPase alpha1A4:GFP strain zebrafish and wt1b:GFP strain zebrafish were selected as broodstock for hybridization. The parental zebrafish have been screened by fluorescence at the embryonic or juvenile stage to ensure that the Na,K-ATPase alpha1A4:GFP strain broodstock exhibits specific expression of green fluorescent protein in the renal tubules and pronephric ducts of the parent fish, and the wt1b:GFP strain Partial expression of green fluorescent protein was found in anterior glomeruli and anterior tubules of broodstock. figure 1 It is the fluorescent picture of the parental Na,K-ATPase alpha1A4:GFP strain zebrafish 48 hours after fertilization; figure 2 is the fluorescent image of the parental wt1b:GFP line zebrafish 48 hours after fertilization.

[0028] S...

Embodiment 2

[0052] The method of the present invention is used to obtain the zebrafish expressing green fluorescent protein in the whole kidney. Its similarities with Embodiment 1 will not be repeated, and its difference is:

[0053] 3. Screening of hybrid traits

[0054] The fertilized eggs were cultured in constant temperature aerated water at 26°C-29°C for 28 hours, and then the expression effect of the green fluorescent protein spot gene was detected using a fluorescence microscope; zebrafish embryos with fluorescence in the anterior glomerulus, tubules, and ducts were selected for hybrid offspring cultivation . Embryos that do not fluoresce or are dead are removed from the dish.

Embodiment 3

[0056] The method of the present invention is used to obtain the zebrafish expressing green fluorescent protein in the whole kidney. The same parts as in Example 1 will not be repeated, and the difference is that the temperature of the culture water at each stage is adjusted from 26° C. to 29° C. to 28.5° C.

technical field

[0001] The invention relates to a zebrafish breeding method, in particular to a breeding method for obtaining whole kidneys expressing green fluorescent protein zebrafish, and belongs to the field of biotechnology. Background technique

[0002] In 1962, Shimomura et al. first discovered the bioluminescent protein aequorin in Aequoria victoria, and observed a by-product of light green fluorescence under ultraviolet irradiation - green fluorescent protein (Green fluorescent protein). Fluorescent Protein, GFP). In 1974, Shimomura et al. completed the purification of GFP, and Prasher et al. cloned its gene 18 years later. In 1994, Chalfie et al. realized the expression of GFP in E. coli cells and Caenorhabditis elegans. As an important biomarker protein, GFP can self-catalyze to form a chromophoric structure and emit green fluorescence under ultraviolet or blue light excitation. Because GFP has many advantages such as stable fluorescence, safety and non-toxicit...

Examples

Embodiment 1

[0025] Such as Figure 1 ~ Figure 4 As shown, the whole kidney expressing green fluorescent protein zebrafish was obtained by the method of the present invention.

[0026] 1. Breeding of hybrid parents

[0027] Na,K-ATPase alpha1A4:GFP strain zebrafish and wt1b:GFP strain zebrafish were selected as broodstock for hybridization. The parental zebrafish have been screened by fluorescence at the embryonic or juvenile stage to ensure that the Na,K-ATPase alpha1A4:GFP strain broodstock exhibits specific expression of green fluorescent protein in the renal tubules and pronephric ducts of the parent fish, and the wt1b:GFP strain Partial expression of green fluorescent protein was found in anterior glomeruli and anterior tubules of broodstock. figure 1 It is the fluorescent picture of the parental Na,K-ATPase alpha1A4:GFP strain zebrafish 48 hours after fertilization; figure 2 is the fluorescent image of the parental wt1b:GFP line zebrafish 48 hours after fertilization.

[0028] S...

Embodiment 2

[0052] The method of the present invention is used to obtain the zebrafish expressing green fluorescent protein in the whole kidney. Its similarities with Embodiment 1 will not be repeated, and its difference is:

[0053] 3. Screening of hybrid traits

[0054] The fertilized eggs were cultured in constant temperature aerated water at 26°C-29°C for 28 hours, and then the expression effect of the green fluorescent protein spot gene was detected using a fluorescence microscope; zebrafish embryos with fluorescence in the anterior glomerulus, tubules, and ducts were selected for hybrid offspring cultivation . Embryos that do not fluoresce or are dead are removed from the dish.

Embodiment 3

[0056] The method of the present invention is used to obtain the zebrafish expressing green fluorescent protein in the whole kidney. The same parts as in Example 1 will not be repeated, and the difference is that the temperature of the culture water at each stage is adjusted from 26° C. to 29° C. to 28.5° C.

Images