CN1590548A - Recombinant plasmid expressing two fluorescent genes - Google Patents

Abstract

The present invention provides a recombinant plasmid comprising: a ubiquitous promoter, (b) a fluorescent gene operably linked and inserted downstream of the ubiquitous promoter, (c) a skin-specific or muscle-specific promoter, and (d) another fluorescent gene operably linked and inserted downstream of the skin-specific or muscle-specific promoter, wherein the ubiquitous promoter and the skin-specific or muscle-specific promoter have reverse directionality, and the ubiquitous promoter and the skin-specific or muscle-specific promoter are respectively located upstream of the fluorescent gene and the another fluorescent gene so as to have directionality for transcription of the genes. In addition, the invention also provides a host cell, a method for producing the transgenic fish and the transgenic fish with the plasmid of the invention.

Description

Recombinant plasmid expressing two fluorescent genes

technical field

The invention relates to a recombinant plasmid and a method for producing transgenic fish, host cells and transgenic animals containing the recombinant plasmid.

Background technique

Transgenic fish research utilizes genes driven by heterologous and homologous regulatory elements and produced by constitutive or tissue-specific expression of genes. Regulatory elements include genes for antifreeze protein, mouse metallothionein, chicken δ-crystal, carp β-actin, salmon histone H3 and carp α-globulin, etc. However, the use of these DNA elements in transgenic fish has significant disadvantages, including low expression efficiency and mosaic expression of the transgene.

Microinjection of the lac reporter gene driven by the mekada β-actin promoter into medaka eggs resulted in transient expression of the lacZ gene, even though expression was minimal and highly mosaic. It is also expressed in the first offspring. Similar results were reported by Hamada et al. in medaka embryos produced by microinjection of green fluorescent protein fused to the medaka β-actin promoter into fish eggs. (Hamada et al., 1998, Mol Marine Biol Biotechnol 7:173-180).

Chi-Yuan Chou et al. published a DNA construction plasmid with inverted terminal repeats linked at both ends to increase the efficiency of transgene expression in medaka fish. The expression of the transgene was consistent in the 0th generation and the next two generations (Chi-Yuan Chou et al., 2001, Transgenic Research 10: 303-315). In addition, Chung-Der Hsiao et al. also pointed out that the insertion of adeno-associated virus inverted terminal repeats (AAV-ITRs) into the transgene will make the gene expression of the 0th generation of zebrafish consistent and make the transfer of the transgene very stable (Chung- Der Hsiao et al., 2001, Developmental Dynamics 220: 323-336).

Zebrafish (Danio rerio) is a new model organism for the study of vertebrate developmental biology. Using zebrafish as an experimental model has several major advantages, such as easy access to eggs and embryos, clear organization of the embryogenesis process, in vitro development, short generation time, and easy rearing of both adults and juveniles.

Type I species including rat myosin light chain enhancers (Moss, J.B. et al., Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish. Gene 173, 8998, 1996), zebrafish and tilapia have been utilized. Insulin growth factor promoter (Chen, J.Y. et al., Isolation and characterization of tilapia (Oreochromismossambicus) insulin-like growth factors gene and proximal promoter region (DNA Cell Biol. 17, 359-376, 1998) and other different gene promoters , introducing known fluorescent genes such as the GFP gene (including the EGFP gene) into zebrafish. The purpose of these transgenic experiments is to develop a GFP transgenic system that can be used to analyze gene expression or to test regulatory DNA in gene promoters element.

Patent application WO0049150 discloses a fluorescent transgenic ornamental fish expressed by a single fluorescent gene (such as GFP). However, any fish that can consistently and stably express two or more fluorescent genes has not yet been developed.

Contents of the invention

The present invention relates to a recombinant plasmid comprising: (a) a ubiquitous promoter, (b) a fluorescent gene operably linked and inserted downstream of the ubiquitous promoter, (c) a skin-specific or a muscle-specific promoter, and (d) another fluorescent gene operably linked and inserted downstream of the skin-specific or muscle-specific promoter, wherein the ubiquitous promoter and skin-specific or muscle-specific Sexual promoters have reverse (adverse directional property), and ubiquitous promoters and skin-specific or muscle-specific promoters are located upstream of the fluorescent gene and the other fluorescent gene, respectively, to have The directionality of transcription.

The invention also relates to a host cell containing the plasmid of the invention.

In addition, the present invention also relates to a method for producing transgenic fish, the method comprising:

a) introducing the plasmid of the present invention into fish egg cells or embryo cells, and

b) Developing egg cells or embryonic cells into fish, wherein the plasmid of the present invention is introduced into the genome of the fish.

The present invention further relates to a method for producing transgenic fish expressing two fluorescent genes simultaneously, the method comprising the following steps:

a) cutting the plasmid of the present invention with a restriction enzyme at an appropriate restriction enzyme cutting position to obtain two plasmid fragments I and II, wherein the plasmid I contains fragments a) and b) as defined in the plasmid of the present invention, and The plasmid II contains c) and d) fragments as defined in the plasmid of the present invention;

b) introducing the plasmids A and B of step a) into fish egg cells or embryo cells respectively; and

c) making the fish express plasmids I and II simultaneously.

The present invention also relates to a transgenic animal transformed with the inventive plasmid.

Description of drawings

Figure 1 shows photographs of transgenic fish expressing two fluorescent genes.

Figure 2 shows a flow chart for the production of plasmid C following ligation of plasmid A and plasmid B.

Detailed ways

The invention provides a recombinant plasmid capable of introducing two or more genes (for example, green and red fluorescence) into fish. Under normal light sources, these fish will show uniform and strong fluorescence.

The present invention provides a recombinant plasmid comprising: (a) a ubiquitous promoter, (b) a fluorescent gene operably linked and inserted downstream of the ubiquitous promoter, (c) a skin-specific or A muscle-specific promoter, and (d) another fluorescent gene operably linked and inserted downstream of the skin-specific or muscle-specific promoter, wherein the ubiquitous promoter and skin-specific or muscle-specific Sexual promoters are reversed, and ubiquitous promoters and skin-specific or muscle-specific promoters are located upstream of the fluorescent gene and the other fluorescent gene, respectively, so as to have directions for transcription of the genes sex.

The terms "upstream" and "downstream" mentioned in the article mean that when the reference direction is defined as the direction from the start codon to the stop codon of the fluorescent gene, a certain point is located at one end in the same direction as the reference direction, which is called The "downstream" of the point, and the opposite end of the reference direction is the "upstream" of the point.

According to the present invention, its ubiquitous promoter is used to drive the plasmid of the present invention to express the fluorescent gene. The ubiquitous promoter and the skin-specific or muscle-specific promoter have inversion, and the ubiquitous promoter is located upstream of the fluorescent gene, so as to have the directionality for transcription of the fluorescent gene. The ubiquitous promoter is preferably selected from the group consisting of β-actin, elongation-1-α, 18S-rDNA or 5S-rDNA.

According to the present invention, skin-specific or muscle-specific promoters are used to drive the expression of fluorescent genes. The skin-specific or muscle-specific promoter is located upstream of the fluorescent gene, so as to have the directionality available for the transcription of the fluorescent gene. The skin-specific or muscle-specific promoter is preferably selected from the group consisting of α-actin, troponin T, troponin C, heavy chain of myosin, cytokeratin type II C II C) or the group consisting of S-100.

According to the present invention, any fluorescent gene can be inserted upstream of the promoter of the plasmid of the present invention. The fluorescent gene is preferably selected from the group consisting of green, red, yellow or blue fluorescent genes. These fluorescent genes are commercially available, for example, from Clonteh Laboratories Inc., Lightools Research, and BD Biosciences Pharmingen, etc. Preferably selected from the group consisting of green and red fluorescent genes.

Hiroshi Otsuki et al. showed that when using a ubiquitous promoter, all parts of rice crops including calli showed green fluorescence (Hiroshi Otsuki, January 12-16, 2002, Plant, Animal & Microbe Genomes X Conference). According to the present invention, the green fluorescent gene can be operably linked and inserted downstream of the ubiquitous promoter of the present invention. Green fluorescent protein (GFP) was originally isolated from a species of jellyfish (Aequorea victoria) and is commercially available. Unlike other bioluminescent reporters, GFP emits bright green light when exposed to ultraviolet or blue light. The emitted green light is due to the energy transfer from aequorin to GFP. GFP is a protein composed of 238 amino acids with a molecular weight of 28kDa. Its spectrum has a main absorption peak at 395nm, a secondary peak at 470nm, and a single emission peak at 509nm. The advantage of GFP is that its fluorescence is not species-dependent, and it can emit green light without relying on substrates, cofactors or other proteins. Several host organisms and cells such as Escherichia coli, yeast, mammalian cells, insect cells and plant cells have been successfully used to express GFP.

According to the present invention, the red fluorescent gene can be purchased from BD Bioscience Clontech. In practicing the present invention, the pDsRed2-1 line is used as the source of the red fluorescent gene. pDsRed2-1 encodes DsRed2, an engineered variant of DsRed with faster maturation and less nonspecific aggregation. DsRed2 is derived from the red fluorescent protein (drFP583; Matz, MV, et al. (1999) Nature Biotech.17:969-973.) of Lentinus edodes coral, which contains a series of silent base pair changes, and the codon usage preference of humans is in High expression in mammalian cells is consistent (Haas, J., et al. (1996) Curr. Biol. 6:315-324.). In mammalian cell culture, when DsRed2 is constitutively expressed, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble protein aggregates frequently observed in bacterial and mammalian cell systems expressing DsRed1 were dramatically reduced in cells expressing DsRed2. DsRed that matures faster and is more soluble also makes host cells more tolerant. Mammalian cell cultures transfected with DsRed2 showed no significant signs of reduced viability, and in those cell lines tested, cells expressing DsRed2 exhibited morphological (e.g. adhesion, light refraction) and growth characteristics comparable to those of untransfected cells. same as the stained control group. pDsRed2-1 is a promoter-free DsRed2 vector that can be used to monitor the transcription of different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). The sequence upstream of DsRed2 was changed to the Kozak consensus translation initiation site (Kozak, M. (1987) Nucleic Acids Res. 15:8125-8148.) to increase translation efficiency in eukaryotic cells. The SV40 polyadenylation signal downstream of the DsRed2 gene directs the proper processing of the 3' end of the DsRed2 mRNA. The vector mainly contains an SV40 origin for replication of mammalian cells expressing the SV40T antigen, a Puc origin of replication for propagation in E. coli, and an f1 origin for making single-stranded DNA. A neomycin resistance cassette (Neo ^r ) can be used to select stably transfected mammalian cells using G418. This cassette contains the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and Polyadenylation signal from the herpesvirus thymidine kinase (HSV TK) gene. In E. coli, the bacterial promoter upstream of the cassette expresses kanamycin resistance.

According to the present invention, recombinant plasmids are constructed by combining a plasmid containing a ubiquitous promoter and a fluorescent gene, and a plasmid containing a skin-specific or muscle-specific promoter and another fluorescent gene using standard molecular techniques in the field. of. When implementing the present invention, the plasmid containing the ubiquitous promoter and the green fluorescent gene was cut with restriction enzymes to obtain a 4.7 kb fragment. The plasmid containing skin-specific or muscle-specific promoter and red fluorescent gene was also cut with restriction enzymes to obtain a 7.5kb fragment. The resulting 4.7kb and 7.5kb fragments were joined together via a ligation step.

The invention provides a host cell containing the plasmid of the invention. According to the present invention, some host systems can be utilized to accommodate the plasmid of the present invention, and these host systems include microorganisms such as bacteria transformed with recombinant phage, plasmids or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, infected with virus expression vectors Insect cell systems, plant cell systems infected with viral expression vectors or bacterial expression vectors, or animal cell systems, etc., but not limited thereto.

The invention provides a transgenic animal transformed with the plasmid of the invention. According to the present invention, the transgenic animal can be any conveniently obtained animal, such as non-human mammals, such as rodents and fish used in laboratory tests as examples. The transgenic animal of the present invention can be obtained conveniently by introducing the plasmid of the present invention into the animal body by conventional and convenient gene manipulation techniques such as microinjection or injection of recombinant vectors. A gene can be introduced directly or indirectly into a cell or all cells of an animal by introduction into a precursor of the cell. Gene manipulation techniques include classical cross-breeding, in vitro fertilization, and the introduction of recombinant DNA molecules that may integrate into chromosomes or become extrachromosomally replicated DNA. The transgenic animal is preferably a transgenic fish, and a transgenic animal selected from medaka, zebrafish, discus, goldfish, squid, cichlid, guppy, arowana, koi, betta or other ornamental fish Fish is more preferred. According to the present invention, the invented transgenic fish can express a color mixed with green and red.

According to one embodiment of the present invention, the plasmid of the present invention can be used to produce fish expressing one kind of fluorescence, doping with fluorescence and expressing different fluorescence at the same time. For expression of a fluorescent gene, the inventive plasmid is cut with appropriate restriction enzymes to generate a plasmid fragment containing a fluorescent gene. The resulting plasmid is introduced into fish egg cells or embryo cells to obtain fish expressing a fluorescent gene. For expression of doped fluorescence, the inventive plasmid is introduced into fish eggs or embryo cells to obtain fish expressing doped fluorescence. As far as expressing different fluorescences at the same time, different plasmids containing different fluorescent genes are introduced into fish eggs or embryo cells to obtain fishes expressing different fluorescent genes at the same time.

Preferably, the plasmid of the present invention can be used to produce fish expressing a single green fluorescent gene, a single red fluorescent gene, doped with green and red fluorescent genes or simultaneously expressing green and red fluorescent genes. For fish expressing green fluorescence, the plasmid of the present invention is cut at an appropriate position with restriction enzymes to obtain a plasmid containing a ubiquitous promoter and a green fluorescent gene operably linked and inserted downstream of the ubiquitous promoter. The resulting plasmid can be introduced into fish eggs or embryos to express green fluorescence. For fish expressing red fluorescence, the plasmid of the present invention is cut at an appropriate position with restriction enzymes to obtain a promoter containing skin-specific or muscle-specific and operatively connected and inserted into the skin-specific or muscle-specific The plasmid of the red fluorescent gene downstream of the promoter, and the resulting plasmid can be introduced into fish egg cells or embryonic cells to express red fluorescent light.

According to a preferred embodiment of the present invention, the present invention provides a method for producing transgenic fish expressing doped fluorescence, the method comprising a) introducing the inventive plasmid into fish egg cells or embryo cells, and b) allowing egg cells or The embryonic cells develop into fish, wherein the invented plasmid is introduced into the genome of the fish to obtain a fish expressing the doped fluorescence. The fluorescent gene is preferably selected from the group consisting of green, red, yellow or blue fluorescent genes, more preferably if the fluorescent gene is selected from the group consisting of green or red fluorescent genes.

According to a preferred embodiment of the present invention, the present invention provides a method for producing transgenic fish that simultaneously express two different fluorescences, the method comprising the following steps:

a) After cutting the plasmid of claim 1 with a restriction enzyme at the appropriate restriction enzyme cutting position, two plasmid fragments I and II can be obtained, wherein plasmid I contains the fragments a) and b) defined in claim 1, and plasmid II contains Fragments c) and d) as defined in claim 1;

b) introducing each plasmid A and plasmid B described in step a) into fish egg cells or embryo cells respectively,

c) making the aforementioned fish express plasmids I and II simultaneously.

According to the present invention, the fish produced by the inventive method can simultaneously express two different fluorescences. The plasmid of the present invention is cut at an appropriate position with a restriction enzyme to obtain two plasmids, one of which contains a ubiquitous promoter and a fluorescent gene operatively linked and inserted downstream of the ubiquitous promoter, and the other plasmid contains A skin-specific or muscle-specific promoter and another fluorescent gene operably linked and inserted downstream of the skin-specific or muscle-specific promoter. Then, the two generated plasmids were separately introduced into fish to each express a different fluorescent gene. It is preferred that the fluorescent gene is selected from the group consisting of green, red, yellow or blue fluorescent genes, and it is more preferred that the fluorescent gene is selected from the group consisting of green and red fluorescent genes.

According to a preferred embodiment of the present invention, the fish obtained from the method of the present invention can simultaneously exhibit red and green fluorescence. Preferably, the fish obtained from the method of the present invention can be further bred and reproduced using techniques known in the art, so as to obtain fishes that consistently and stably express red and green fluorescence.

The following examples further illustrate the invention, but are not intended to limit the scope of the invention.

Example

Example 1 Construction of a plasmid comprising a GFP sequence

Isolation of skin- or muscle-specific clonal strains that ubiquitously express zebrafish cDNA. Isolation and sequencing of cDNA clones were described by Gong, Z. et al., Gene 201, 87-98 (1997). Basically, random cDNA clones were selected from cDNA gene banks of zebrafish embryos and adults, and each cDNA clone was partially sequenced through a single sequencing reaction. This partial sequence was then used to confirm the potential function and tissue specificity of the sequenced clones.

As shown in Figure 2, plasmid A is a structural view of a constructed plasmid. GFP is driven by a ubiquitous promoter. After the plasmid was linearized by NotI and PstI restriction enzymes, its cohesive ends were blunted with deoxynucleotide (dNTP), and a 4.7 kb fragment was recovered from agarose gel electrophoresis. The composition of this plasmid is constructed according to the description of the present invention.

Example 2 Construction of a plasmid containing the red fluorescent protein sequence

As in Example 1, plasmid B is a structural view of the constructed plasmid as shown in FIG. 2 . DsRed is driven by a skin-specific or muscle-specific promoter. After the plasmid was linearized with PstI restriction enzyme, its cohesive ends were blunted with deoxynucleotide (dNTP), and a 7.5 kb fragment was recovered from agarose gel electrophoresis. The composition of this plasmid is constructed according to the description of the present invention.

Example 3 Construction of plasmids by connecting plasmids A and B

As shown in FIG. 2 , plasmid C is a structure for constructing a plasmid, expressed as a linearized fragment cleaved by restriction enzyme NotI, or a linearized fragment cleaved by restriction enzyme NotI or SalI. This plasmid illustrates the composition of the construction plasmid for gene transfer according to the present invention.

Example 4 Introduction of plasmids and fluorescence microscopy

Zebrafish were reared under artificial conditions of fourteen hours of light and ten hours of darkness with Tetramin (TetraGermany). Eggs were collected within thirty minutes of fertilization and the connecting filaments were removed. Fertilized eggs were kept at 6°C until microinjection with DNA fragments (such as linearized plasmid C cut with NotI or linearized plasmid C cut with NotI and SalI) before the first division, at 10 μg /ml concentration into the cytoplasm. Injected eggs were incubated in distilled water at 26°C. Embryos were observed with a dissecting stereomicroscope (MZAPO, Leica, Germany) under bright field. Detection of dark field luminescence for green and/or red fluorescence was performed with a stereomicroscope equipped with GFP and/or red fluorescent protein. Photographs were taken with a camera equipped with ISO 400 film and a controller for film timed exposure.

Example 5: Production of transgenic zebrafish expressing two types of fluorescence

zebrafish reproduction

A pool of male and female zebrafish was housed in a 60×20×30 cm glass aquarium with a temperature of 28.5° C. and a photoperiod of 14 hours. The fish were fed twice a day with Artemia worms, after which several pairs of strong male and female zebrafish were selected and placed in a 30 × 10 × 20 cm incubation tank with a net for collecting eggs. To establish transgenic strains, a pair of transgenic fish and wild species were reared in 22 × 14 × 13 cm tanks.

Gene transfer and selection of original transgenic fish (transgenic founder)

Fertilized eggs were collected in plastic capillary tubes and placed in racks. A glass needle with an opening of 10 μm was filled with linearized plasmid solution and fixed with mineral oil. A volume of 2-4 nl of the DNA sample is then microinjected into single-cell fertilized eggs. The injected fertilized eggs were hatched in a petri dish containing a low concentration of methylene blue solution, and placed in an incubator set at 28°C. On the third day, embryos expressing fluorescence can be observed using a fluorescence microscope. Five days later, the fluorescent zebrafish were moved to a fish tank for rearing. Sexual maturity can be reached after 12 weeks.

Generation of germline transmission of fluorescent transgenic zebrafish

Putative stock transgenic fish with fluorescent expression were crossed with wild strains. Transgenic F2 (second generation) is obtained by crossing two fluorescent F1s, and the expression of fluorescence can be observed throughout the life of this fish.

Example 6: Potential applications of fluorescent transgenic fish

Fluorescent transgenic fish are commercially available as ornamental fish. Stable transgenic strains can be developed by mating GFP transgenic animals with wild fish or another transgenic fish. A wider variety of fluorescent transgenes can be produced by isolating more zebrafish gene promoters, e.g., eye-specific, bone-specific, tail-specific, etc., and/or raising these transgenic zebrafish by traditional methods zebrafish.

Because the blue fluorescent protein (BFP) gene, yellow fluorescent protein (YFP) gene and cyan fluorescent protein (CFP) gene are all commercially available at Clonetech, fluorescent fish of various colors can be produced using the same technology. For example, transgenic fish with GFP under an eye-specific promoter, BFP under a skin-specific promoter, and YFP under a muscle-specific promoter will appear Fluorescent colors in the following varieties: green eyes, blue skin, and yellow eyes. After recombining promoters with different tissue specificities and fluorescent protein genes, more transgenic fish with different color changes can be created. An intermediate color can be created by using the same tissue to express two or more different fluorescent proteins.

By using promoters inducible by heavy metals (e.g. cadmium, cobalt, chromium) or hormones (e.g. estrogen, androgen, or other steroid hormones), it is possible to develop a method for monitoring environmental pollution and evaluating human drinking and hydroponic use. Bio-sensing system for water quality. In such a biosensing system, when pollutants such as heavy metals and estrogens (or their derivatives) reach a threshold concentration in the aquatic environment, the transgenic fish will emit green fluorescence (or other colors, depending on Depending on the fluorescent protein gene used). This biosensing system is superior to traditional analytical methods because it is fast, visible, and can directly identify specific compounds in complex mixtures found in aquatic environments, and it is portable or requires fewer accessories. instrument. In addition, biosensing systems can also provide direct information on biological toxicity and are biodegradable and renewable.

Environmental monitoring of several substances can also be done by creating a transgenic fish with a gene that encodes fluorescent proteins of different colors driven by promoters that respond to various substances, and then observing the fish in the environment specific colors shown in . Alternatively, some fish can be transformed with a unique vector, which can then be incorporated into a school of fish used to monitor the environment and observe the color expressed by each individual fish.

In addition, fluorescent transgenic fish should also have value in the market for scientific research materials, as they can be used for studies such as tracking cell lines and embryos for cell migration. Cells from transgenic fish expressing GFP can also be used as cells and genetic markers in cell transplantation and nuclear transfer experiments.

The present invention has successfully demonstrated that two fluorescent genes can be constructed in zebrafish, which can also be used in other species of fish, such as medaka, goldfish, carp including koi, mahi, tilapia, kiss Larvae, catfish, angel fish, discus, eel, betta, goby, vest, guppy, swordtail (red sword), hatchet, molly, freshwater shark, etc.

The above descriptions are only descriptions of some preferred embodiments of the present invention, so all changes that apply the equivalent methods of the description and claims of the present invention should be included in the patent scope of the present invention.

Claims (12)

1. recombinant plasmid, comprise: (a) ubiquitous promotor, (b) fluorogene, this gene can be controlled the downstream that connects and insert this ubiquitous promotor, (c) promotor of skin specificity or muscle specific, and (d) another fluorogene, this gene can be controlled the downstream that connects and insert this skin specificity or muscle specific promotor, wherein this ubiquitous promotor and skin specificity or muscle specific promotor have reverse property, and this ubiquitous promotor and skin specificity or muscle specific promotor lay respectively at the upstream of this fluorogene and this another fluorogene, can be for the directivity of described genetic transcription to have.

2. the recombinant plasmid of claim 1, the wherein group formed of the optional free beta-actin of this ubiquitous promotor, elongation-1-α, 18S-rDNA or 5S-rDNA.

3. the recombinant plasmid of claim 1, the wherein optional free α-Ji Dongdanbai of the promotor of this skin specificity or muscle specific, troponin (troponin) T, TnC, myosin heavy chain, Cytokeratin II C type (cytokeratin type II C) or group that S-100 formed.

4. host cell, it comprises the plasmid of claim 1.

5. method that produces genetically engineered fish, this method comprises:

A) plasmid with claim 1 imports in fish egg cell or the embryonic cell, and

B) make this ovum or embryonic cell grow adult fish, wherein the plasmid of claim 1 imports in the genome of this fish.

6. the genetically engineered fish of claim 5, the wherein group formed of the optional free medaka of this fish, zebra fish, seven color angle fish, goldfish, Medaka fish, kind porgy, peacock fish, imperial fish, carp or bucket fish.

7. genetically engineered fish, it comprises (a) ubiquitous promotor, (b) fluorogene, this gene can be controlled the downstream that connects and insert this ubiquitous promotor, (c) skin specificity or muscle specific promotor, and (d) another fluorogene, this gene can be controlled the downstream that connects and insert this skin specificity or muscle specific promotor, wherein this ubiquitous promotor and skin specificity or muscle specific promotor have reverse property, and this ubiquitous promotor and skin specificity or muscle specific promotor lay respectively at the upstream of this fluorogene and this another fluorogene, can be for the directivity of described genetic transcription to have.

8. the genetically engineered fish of claim 7, wherein this fluorogene is selected from the group of being made up of green, red, yellow or blue-fluorescence gene.

9. the genetically engineered fish of claim 8, wherein this fluorogene is selected from the group of being made up of green and red fluorescence gene.

10. the method for the genetically engineered fish of two kinds of different fluorogenes is expressed in a manufacturing simultaneously, and this method comprises the following step:

A) at suitable restriction enzyme cleavage site, plasmid with restriction enzyme cutting claim 1, to obtain two plasmid fragment I and II, wherein this plasmid I contain claim 1 defined a) and b) fragment, plasmid II then contains the defined c of claim 1) and d) fragment;

B) respectively the plasmid I of this step a) and II are imported in fish egg cell or the embryonic cell, and

C) make this fish give expression to plasmid I and II simultaneously.

11. the method for claim 10, the wherein group formed of optional free green, red, yellow of this fluorogene or blue-fluorescence gene.

12. the method for claim 11, the wherein group formed of optional free green of this fluorogene or red fluorescence gene.

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