CN1647625A - Method for producing new fluorescent fish and new fluorescent fish produced by the method - Google Patents

Abstract

The present invention relates to a method for producing new ornamental fish by breeding transgenic fish with fish having different phenotypes or behavioral patterns. The new transgenic fish obtained have a phenotype or behavioral pattern that is different from either parent. The invention also provides novel transgenic fish produced by the method.

Description

Method for producing new fluorescent fish and new fluorescent fish produced by the method

technical field

The invention relates to a method for producing a new ornamental fish and a new ornamental fish produced by the method.

Background technique

Transgenic technology has become an important tool for studying gene function today. This technique was first developed in mice. After the researchers injected foreign DNA into fertilized eggs, they found that some mice grown from the injected fertilized eggs retained the foreign DNA.

Ornamental fish is a fish industry with a global market. Therefore, it is of great commercial value to use recombinant DNA technology and transgenic technology to modify the appearance of ornamental fish. Many techniques for introducing exogenous genes into fish have been developed, including microinjection, electroporation, sperm-mediated gene transfer, gene gun, liposome-mediated Guided gene transfer and direct injection of DNA into muscle tissue (Xu et al., DNA Cell Biol. 18, 85-95 (1999)).

Genetically modified fish have been produced for recreational use. For example, the fluorescent fish expressing GFP fluorescence is produced by injecting the GFP gene connected with the fish specific gene promoter (promoter) into fertilized eggs (Hamada, K., etc., Mol.Marine Biol.Biotech., 7, 173-180 (1998)). Tanaka et al. produced transgenic medaka fish with green fluorescence only in germ cells (Tanakal et al., 22nd Annual Meeting of the Molecular Biology Society of Japan Brief pp. 458 (1999)). However, it is obvious that using transgenic technology to produce ornamental fish with different fluorescent colors, different appearances or behavior patterns one by one is very time-consuming, laborious and expensive.

One of the goals of the farmed fish industry is to use genetic changes to produce improved fish species. The genome of the progeny fish is the combination of the genomes of the parental male and the female gamete, which forms an embryo through fusion, and then becomes a progeny fish. Therefore, crossing transgenic fish with the same or different species of fish with different phenotypes or behavioral patterns, such as fish with different markings, stripe colors, or environmental tolerance, can provide ornamental fish in a faster and cheaper way than producing transgenic fish one by one. In addition to more options, these fish are beyond the reach of existing genetically modified fish technology.

Contents of the invention

The present invention provides a method for producing new ornamental fish, which comprises

(a) producing a transgenic fish containing one or more linked fluorescent genes;

(b) crossbreeding the transgenic fish with fish of a different phenotype or behavioral pattern; and

(c) Screening for transgenic offspring having a different phenotype or behavioral pattern than the parent.

The present invention also provides novel transgenic fish produced by this method.

Description of drawings

Figure 1 reveals the plasmid map (a) pβ-actin-EGFP; (b) pβ-actin-EGFP-ITR.

Fig. 2 discloses the construction process of plasmid pβ-DsRed2-1-ITR prepared from plasmid pOBA-109 and pDsRed2-1-ITR.

Fig. 3 reveals the p-αDsRedITR plasmid construction diagram, including the inserted gene fragment and restriction enzyme cutting site.

Figure 4 shows breeding of red TK-1 to Hong Kong medaka, breeding of green TK-1 to Hong Kong medaka, breeding of red TK-2 to colorful leopard zebra and breeding of red TK-2 to leopard The zebra is mated, the process of mating the red TK-2 with the baby zebra.

Figure 5 reveals pictures of (a) red TK-1, (b) Hong Kong medaka and (c) red TK-1 x Hong Kong medaka.

Figure 6 reveals pictures of (a) green TK-1; (b) Hong Kong medaka fish and (c) green TK-1 x Hong Kong medaka fish.

Figure 7 reveals pictures of (a) red TK-2; (b) colorful leopard zebra and (c) red TK-2×colorful leopard zebra.

Figure 8 reveals pictures of (a) red TK-2; (b) leopard zebra and (c) red TK-2 x leopard zebra.

Figure 9 reveals pictures of (a) red TK-2; (b) baby zebra and (c) purple fluorescent fairy.

Detailed ways

The present invention provides a method of producing a new ornamental fish having a different phenotype or behavioral pattern from its parent. The present invention also provides novel ornamental fish produced by this method.

The ornamental fish used herein refers to transgenic fish or its progeny fish containing exogenous gene carrier. Fish containing the gene carrier include fish grown from embryos containing the gene carrier. The exogenous gene carrier used herein refers to the artificial introduction of nucleic acid into fish, or the initial artificial introduction into fish. The term artificial introduction is intended to exclude the introduction of a gene carrier via ordinary mating. That is, genes that were originally introduced into the animal strain via mating houses were excluded. However, the fish that obtained the exogenous gene carrier by transferring (generally mating) from the fish containing the gene carrier (that is, the gene carrier was initially introduced artificially) is considered to contain the exogenous gene carrier. This fish is the offspring of the fish into which the foreign gene carrier was introduced. As used herein, the offspring of a fish is any fish that reproduces via sexual reproduction or cloning and inherits the genetic material of the parent. In this context, cloning refers to the production of a genome-identical fish from DNA or from a single fish cell or from multiple fish cells. The line that can reproduce other fish is called progenitor fish. As used herein, the process of growing from single or multiple cells (such as embryonic cells) to fish refers to the growth process of growing, dividing and differentiating from fertilized eggs or embryonic cells (and their progeny cells) into adult fish.

The following examples illustrate how offspring fish with different phenotypes and behavioral patterns can be produced from parent fish and their uses. The transgenic fish described in the examples are particularly fruitful for increasing the commercial value of ornamental fish.

transgenic vector

The transgenic vector is the genetic material introduced into the fish to produce the transgenic fish. These gene carriers are artificially introduced into fish. The way of introduction and the structure of commonly used gene carriers need to be considered as exogenous gene carriers. Although the transgenic vector can be composed of any nucleic acid sequence, the preferred transgenic vector for the transgenic fish disclosed in the present invention functionally links the expression sequence to a sequence that encodes the expression product. The preferred transgenic vector also includes other parts that can help gene expression, stabilize or allow the insertion of the gene vector into the genome of the fish. The part of the transgenic vector used herein refers to a functional or operably connected part so that the two parts can function simultaneously after being connected.

fish species

The gene vectors and methods disclosed here are applicable to all kinds of fish. Fish as used herein refers to any animal taxonomically belonging to the class pisces. Preferred fish are scientifically or commercially useful fish species including Cichlidae (such as Pseudotropheus, Cichlasoma, Apistogramma, Pterophyllum, Symohysodon), Catfishes (such as Corydoras, Ancistrus, Pterygoplichthys), Fighting Fishes (such as Betta, Macropodus), Garasenko ( Characidae) (such as Tetras or Carnegiella), Cyprinidae (such as Cyprinus, Brachydanio, Danio, or Carassius), killifishes ( Such as Medaka, Rivulines, Livebearing Toothcarps).

Preferred fish species are medaka and zebrafish (Brachydanio or Danio). A great advantage of medaka and zebrafish as ornamental fish is that they are both mostly transparent (Kimmel, Trends Genet 5:283-8 (1989)). General zebrafish culture and care are described in detail in Streisinger, Natl. Cancer Inst. Monogr. 65:53-58 (1984). Killifish can be selected from the following species: Oryzias javanicus, Oryzias latipes, Oryzias nigrimas, Oryzias luzonensis, Oryzias profundicola, Oryzias matanensis, Oryzias mekongensis, Oryzias minutillus, Orynotzias melastigma, Hong Kong medaka (Oryzias.bleus.curvicens) oophorus or X. saracinorum. The best species is Oryzias latipes.

Preferred zebrafish may be selected from D. acrostomus, D. aequipinnatus, D. malabaricus, D. albolineatus, D. annandalei, D. apogon, D. apopyris, D. assamensis, D. choprae, D. chrysotaeniatus, D. dangila , D.devario, D.fangfangae, leopard zebra (D.frankei), D.fraseri, D.gibber, D.interruptus, D.kakhienensis, D.kyathit, D.laoensis, D.leptos, D.maetaengensis, D. malabaricus, D. naganensis, D. neilgherriensis, D. nigrofasciatus, D. pathirana, D. regina, D. rerio, D. roseus, D. salmonata, D. shanensis or D. spinosus. The best one is the little zebra.

Medaka and zebrafish embryos are easy to manipulate and nearly transparent. Because of these properties, transgenic medaka or zebrafish eggs or embryos can be used as a system for real-time in vivo identification of successful transgenes when carrying expressible reporter genes and systemic expression sequences. Other fish species having some or all of the same properties are also preferred.

production of parent fish

The transgenic fish disclosed in the present invention is obtained by introducing a transgene vector into fish cells, preferably embryo cells, most preferably single-cell embryos. After the transgenic vector is introduced into the embryonic cells, the embryonic cells can be grown to obtain transgenic fish. It is possible to introduce the transgenic vector into the embryonic cells of the fish, and then directly obtain the transgenic fish from the embryonic cells because most of the fish embryos are grown outside the parent body.

The transgenic vector disclosed in the present invention can be introduced into embryonic cells in any suitable way. Many of these methods have been used in animals and fish. These methods include microinjection, electroporation, particle gun, and methods utilizing liposomes. The preferred way is to inject the transgene vector into fish embryo cells by microinjection.

Embryos and embryonic cells can be collected after delivery. Depending on the species, it is generally preferable to collect fertilized eggs. Fertilization can usually be accomplished by placing a male and a female in a water environment that stimulates mating. After egg collection, the preferred method of introducing the carrier is to first remove the chorion of the eggs. After the chorion is removed, it can be removed manually, or preferably by adding pronase. An egg cell before the first cell division is a one-celled embryo, and this fertilized egg can be identified as an embryonic cell.

After the transgenic vector is introduced into the embryo, the embryo can be cultured into adult fish. This process usually only involves keeping the embryos in the same environment as the eggs. However, embryonic cells can also be cultured briefly in isotonic buffered saline. Gene expression of the transgene can be seen in embryos, where possible.

Fish carrying the transgene can be identified in any suitable manner. For example, the genetic genome of a fish containing a portion of the transgene can be discriminated with appropriate probes. To identify fish expressing the transgene, it is necessary to analyze its expression product. Several techniques are available to detect expression products and can be applied to transgenic fish. Preferably, Northern blotting or Southern blotting is used to detect whether the possible transgenic fish has the sequence of the transgene carrier. Another preferred method is to use PCR or other detection methods for the amplification of specific nucleic acid sequences. Transgenic medaka or zebrafish carrying fluorescent transgenes can be detected directly with the naked eye, as will be described in the examples.

Production of new genetically modified fish

The present invention relates to a method of producing haploid cells and methods of producing novel transgenic fish from these cells.

Preferred haploid cells according to the invention are from meiosis. Meiosis is the first process by which organisms increase genetic diversity. At the same time, it is also the conversion process between diploid and haploid. A specialized cell of the female reproductive system, the oocyte, undergoes meiosis and is embedded within the differentiated oviduct in the ovary. Oocytes undergo meiosis to produce haploid eggs.

In males, haploid spermatozoa are produced through a similar process.

Although there are significant differences in the process of cells becoming male and female gametes, the events that occur in cells during male and female meiosis are very similar, presumably involving common gene products.

The end products of meiosis are haploid cells with different genetic bodies of 4, which undergo cell division to form gametes. The gametes fuse to become zygotes, which grow into embryos and offspring.

To produce new transgenic fish, parental fish carrying the transgene are bred to other parental fish with a different phenotype or behavioral pattern. The phenotype used herein is selected from the group consisting of: color, fluorescence color, fluorescence brightness, fluorescence excitation wavelength, fluorescence distribution pattern, size, body shape, transparency, spot color, spot distribution, stripe color, stripe distribution, number of antennae, antennae type, fin number, fin type, fin size, fin color, tail type, tail size, tail color, eye color, eye type, or any other phenotype that is visibly different from its transgenic counterpart.

Behavioral patterns herein are selected from the group consisting of: odor, fecundity, aggression, nutritional requirements, swimming pattern, growth rate, feed requirements, circadian cycle, lifespan, phototaxis or back-photorotation, or tolerance to the environment.

Haploid gametes from transgenic fish are bred with haploid gametes from parental fish that differ in phenotype or behavioral pattern. Fertilization can be accomplished by any of the known fertilization techniques. Depending on the species of fish, different breeding methods may be required. Methods for producing novel transgenic medaka and zebrafish will be described in the Examples.

The bred transgenic fish may be of the same or different family, genus or species as the parent fish with a different phenotype or behavioral pattern.

Preferred fish species for use in this method are medaka and zebrafish (Brachydanio or Danio). The medaka may be selected from the group consisting of Oryzias javanicus, Oryzias latipes, Oryzias nigrimas, Oryziasluzonensis, Oryzias profundicola, Oryzias matanensis, Oryzias mekongensis, Oryzias minutillus, Oryzias melastigma, Hong Kong medaka, O. celebensis or X. arasphorus. The most preferred species is Oryzias latipes.

Preferred zebrafish may be selected from D. acrostomus, D. aequipinnatus, D. malabaricus, D. albolineatus, D. annandalei, D. apogon, D. apopyris, D. assamensis, D. choprae, D. chrysotaeniatus, D. dangila , D.devario, D.fangfangae, leopard zebra, D.fraseri, D.gibber, D.interruptus, D.kakhienensis, D.kyathit, D.laoensis, D.leptos, D.maetaengensis, D.malabaricus, D. .naganensis, D.neilgherriensis, D.nigrofasciatus, D.pathirana, D.regina, small zebra, D.roseus, D.salmonata, D.shanensis or D.spinosus. Most preferred is a small zebra.

In addition to ornamental purposes, the new transgenic fish in the present invention can be widely used in medical research or research in other life science fields, such as cell fusion, cloning, nuclear transfer, cell apoptosis, cell information and embryonic development research.

Example

The following examples are used to illustrate but not limit various aspects and features of the present invention.

Example 1 Production of plasmid pβ-actin (actin)-EGFPITR

For the production of green fluorescent medaka fish, Green TK-1, we produced the plasmid pβ-actin-EGFPITR. Plasmids of the present invention have been described in detail in Zhou et al., Transgenic Res. 200110(4):303-15 and attached to FIG. 1 . Plasmid pβ-actin-EGFP consists of medaka fish β-actin promoter linked to hGFP1 cDNA, an intron of small t antigen, SV40 polyA, and polyA of the fish β-actin gene. The medaka β-actin promoter was derived from the plasmid pOBA-hGFP1 and digested with the restriction enzymes SalI and Nco. This 3.8 kb fragment was then ligated to a 4.2 kb SalI-NcoI fragment digested from pCMV-EGFPITR. The final product was an -8kb pβ-actin-EGFPITR plasmid, in which the EGFP cDNA was activated by β-actin and had AAV inverted terminal repeats (ITRs) at both ends.

An appropriate amount of plasmid pβ-actin-EGFPITR was digested with NotI. The length of the plasmid pβ-actin-EGFPITR DNA fragment was 7.6 kb as expected.

Example 2 Production of plasmid pβ-DsRed2-1-ITR

For the production of red fluorescent medaka fish, Red TK-1, we generated the plasmid pβ-DsRed2-1-ITR. As shown in Figure 2, the medaka β-actin gene promoter was obtained from the digestion of plasmid pOBA-109 with restriction enzymes NcoI and EcoRI. Firstly, it was digested with restriction enzyme NcoI, then the ends were filled in, and then a 4kb fragment was cut out with restriction enzyme EcoRI.

As shown in Fig. 2, the CMV promoter was excised from the plasmid pDsRed2-1-ITR with restriction enzymes BamHI and SalI. A 4.7 kb fragment was cut out with restriction enzymes BamHI and SalI, and then the medaka β-actin gene promoter was inserted into the place where the CMV promoter was cut out from the plasmid pDsRed2-1-ITR. The obtained plasmid has two ITRs of 145 bp. One ITR is at the 3' end of SV40 poly A. The other is at the 5' end of the promoter of the β-actin gene.

The obtained plasmid pβ-DsRed2-1-ITR, as shown in Fig. 2, has a full length of 8.7 kb. Plasmid pβ-DsRed2-1-ITR includes (1) medaka fish β-actin gene promoter (which induces systemic expression); (2) sea coral red fluorescent protein gene; (3) ITR; and (4) The pUC plasmid of the subject.

An appropriate amount of plasmid pβ-DsRed2-1-ITR was digested with Not I. A small portion of the digested fragment was electrophoresed on an agarose gel to confirm that the fragment was linear. The fragment length was 8.7 kb as expected.

Example 3 Production of plasmid p-αDsRedITR

For the production of red fluorescent zebrafish, Red TK-2, we generated the plasmid pα-DsRed2-1-ITR. As shown in FIG. 3 , the α-actin gene promoter of golden zebrafish comes from digesting plasmid p-aEGFPITR with restriction enzymes NcoI and SalI. Firstly, the restriction enzyme NcoI was used to fill in the ends, and then a 3.8kb fragment was cut out with the restriction enzyme SalI.

As shown in FIG. 3 , the CMV promoter was excised from the plasmid pDsRed2-1-ITR with restriction enzymes BamHI and SalI. First use the restriction enzyme BamHI, then fill in the ends, and then cut out a 4.2 kb fragment with the restriction enzyme SalI. Then the α-actin gene promoter of golden zebrafish was inserted into the place where the CMV promoter was excised from the plasmid pDsRedITR. The obtained plasmid has two ITRs of 145 bp. One ITR is at the 3' end of SV40polyA. The other is at the 5' end of the α-actin gene promoter.

The full length of the obtained plasmid p-αDsRedITR as shown in FIG. 3 is 8.0 kb. The final plasmid pα-DsRedITR includes (1) the α-actin gene promoter of golden zebrafish (which can trigger systemic expression); (2) the red fluorescent protein gene of sea coral; (3) ITR; and (4) The main pUC plasmid.

An appropriate amount of plasmid pα-DsRedITR was digested with Not I. A small portion of the digested fragment was electrophoresed on an agarose gel to confirm that the fragment was linear. The fragment length was 8kb as expected.

Example 4 Preparation of DNA for Microinjection

All DNA plasmids were subjected to ultra-centrifugation in cesium chloride and ethidium bromide gradients (Radloff et al., 1967 Proc Natl Acad Sci USA 57:1514-1521). All DNA fragments to be used for microinjection were precipitated from the agarose gel after electrophoresis.

Example 5 Cytoplasmic Microinjection

The procedure for cytoplasmic microinjection is described in detail in Kinoshita and Ozato, 1995 Fish Biol J MEDAKA 7:59-64, and Kinoshita et al., 1996 Aquaculture 143:267-276.

We reared the fish in an artificial diurnal cycle (14 h light and 10 h dark) at a constant temperature of 26°C and provided a tropical fish flake diet (Tetramin. Tetra, Germany). Fish were collected and separated with divider nets before the vivariums went into a dark cycle. After entering the daylight cycle the next morning, collect the eggs every 15-20 minutes.

The eggs were collected within 30 minutes after fertilization and the attached fibers were removed. The linearized carrier was dissolved in 5X PBS and phenol red (phenol red) and then quantitatively diluted to an applicable concentration. The DNA fragments were aspirated with the microcapillary of a zebrafish microinjector (Drummond), and the diameter of the microcapillary was about 10 μm. When the microcapillary enters the cytoplasm, inject 2-3 nl of DNA solution. A total of 30-40 eggs were injected in each microinjection unit time, and a total of 250-300 eggs were injected in each experiment.

Example 6 Incubation and Screening of Transgenic Embryos

The injected eggs were rinsed with sterile water and placed in an incubator at 28.5°C for cultivation. Embryo fluorescence can be observed within 24 hours. Embryos were observed with a dissecting stereomicroscope (MZAPO, Leica, Germany) under bright field. Dark field luminescence to detect green fluorescence was performed with a stereomicroscope equipped with a GFP Plus filter (480 nm). Place the microinjected embryos into a dish with water. The intensity and distribution of red fluorescence were observed with a fluorescence microscope (Leica MZ-12; Fluorescence System: light source mercury 100W; main emission wavelength 558nm, main absorption wavelength 583nm, filter RFP-Plus). Photographs were taken with an MPS60 camera (Leica, Germany) with an ISO 400 film and a film timing exposure controller. In order to detect the expression distribution of GFP and RFP in transgenic fish, we dissected a larva with green or red fluorescent expression 11 days after fertilization and observed it with a fluorescent microscope. Larvae were fixed at 4°C in 4% paraformaldehyde for 30 minutes, embedded in cryomatrix (Shandon, USA) and frozen at -20°C. 15 μm thick Cryostat sections (Cryostat Microtome, HM500 OM, Microm, Germany) were placed on slides and immediately observed for GFP or RFP fluorescence.

Embodiment 7 produces new transgenic fish

As shown in Figure 4, red TK-1 (Figure 5(a)) was mated with Hong Kong medaka (Figure 5(b)), green TK-1 (Figure 6(a)) was mated with Hong Kong medaka ( Figure 6(b)) was bred, the red TK-2 (Figure 7(a)) was bred with the colorful leopard zebra (Brachydanio sp.) (Figure 7(b)), and the red TK-2 (Figure 8( a)) was bred with leopard zebra (Fig. 8(b)), and red TK-2 (Fig. 9(a)) was bred with baby zebra (Fig. 9(b)).

The F1 progeny transgenic fish produced were red TK-1 × Hong Kong medaka (Fig. 5(c)), green TK-1 × Hong Kong medaka (Fig. 6(c)), red TK-2 × colorful The leopard zebra (Fig. 7(c)), the red TK-2×leopard zebra (Fig. 8(c)) and the purple fluorescent fairy (Fig. 9(c)) are the result of changes in their parental gene combinations. In these examples, fish from one parent express green or red fluorescence, while fish from the other parent have a distinctive distribution of stripes or spots. Therefore, the new transgenic fish produced by the present invention inherits the appearance characteristics of the two parental fishes, but is a new transgenic fish whose phenotype or behavior pattern is different from any parental fish.

The new transgenic fish were then self-crossed to produce homozygous F2 transgenic fish. Thus all offspring of homozygous F2 transgenic fish will carry both the transgene and the new phenotype or behavioral pattern.

Although the present invention has been described and exemplified in detail so that those skilled in the art can produce and use the present invention, various variants, extensions or improvements of the present invention obviously do not go beyond the spirit of the present invention and are included in the scope of the present invention middle.

It will be apparent to those skilled in the art that the present invention is fit for purpose and has the advantages already mentioned, as well as extended advantages. The embryos, animals and methods in the examples are for illustrative purposes only and do not limit the scope of the present invention. Modifications or other uses of the invention may readily be found by those skilled in the art. These changes are also included in the spirit of the present invention and clearly defined in the application scope of the present invention.

Inventions not described in detail herein may be practiced without certain elements or limitations. The terms and expressions used are for the purpose of description rather than limitation, and these terms are not intended to exclude other substitutes which may have the same meaning in the present invention. The inventors recognize that various modifications are possible within the scope of the invention. Therefore, it is particularly emphasized that although the present invention has been disclosed with certain features and certain preferred embodiments, those skilled in the art may resort to modifications or extensions consistent with the spirit of the invention, and these modifications and extensions are still included in this disclosure. The scope of the invention is within and may be defined by the claims appended hereto.

Other embodiments are described in the following claims.

Claims (24)

1. method that produces pet fish, this method comprises:

A) produce a genetically engineered fish, it carries one or more fluorescence protein gene;

B) this genetically engineered fish is bred with the fish with different phenotypes or behavior pattern; And

C) screening has genetically modified filial generation, and described filial generation has phenotype and the behavior pattern that is different from arbitrary parental generation.

2. the process of claim 1 wherein that described fluorescence protein gene is selected from the indigo look fluorescin of the yellow fluorescence protein of the blue fluorescent protein of the red fluorescent protein of the green fluorescent protein of the green fluorescent protein of green fluorescent protein, modification, enhancing, red fluorescent protein, enhancing, blue fluorescent protein, enhancing, yellow fluorescence protein, enhancing, indigo look fluorescin or enhancing.

3. the method for claim 2, wherein said fluorescence protein gene are selected from green fluorescent protein, modify green fluorescent protein, strengthen green fluorescent protein, red fluorescent protein, enhancing red fluorescent protein, blue fluorescent protein or strengthen blue fluorescent protein.

4. the method for claim 3, wherein said fluorescence protein gene are selected from green fluorescent protein, modify green fluorescent protein, strengthen green fluorescent protein, red fluorescent protein or strengthen red fluorescent protein.

5. the process of claim 1 wherein that described genetically engineered fish can be identical or different section, genus or kind with the fish with different phenotypes or behavior pattern.

6. the method for claim 5, wherein said genetically engineered fish and the fish with different phenotypes or behavior pattern can be and not belong to together or not of the same race.

7. the method for claim 6, wherein said genetically engineered fish can be not of the same race with the fish with different phenotypes or behavior pattern.

8. the process of claim 1 wherein described phenotype be selected from color, iridescent, fluorescent brightness, fluorescence exciting wavelength, fluorescence distribution mode, size, build, transparency, spot look, spot distribution, striped look, striped distribution, antenna number, antenna type, fin number, fin type, fin size, fin look, tail type, tail size, tail look, wink, ocular form or other as seen with the different phenotype of its transgenosis pairing person.

9. the process of claim 1 wherein that described behavior pattern is selected from smell, fertility, aggressiveness, nutritional need, swimming mode, growth rate, feed demand, daily cycle, life-span, light or the back of the body optical activity of becoming or to the tolerance of environment.

10. the method for claim 9, wherein said behavior pattern are selected from fertility, swimming mode, growth rate, life-span or to the tolerance of environment.

11. the method for claim 5, wherein said fish are selected from kind porgy, catfish, bucket fish, add Racine section, Cyprinidae, Medaka fish or zebra fish.

12. the method for claim 11, wherein said kind porgy are selected from African kind porgy, the kind porgy in South America, short porgy, angle or seven color angles.

13. the method for claim 11, wherein said bucket fish are selected from bucket fish or color rabbit.

14. the method for claim 11, wherein said catfish is selected from mouse fish, Chinese lute mouse or Pterygoplichthys.

15. the method for claim 11, the wherein said Racine section that adds is selected from lamp fish or axe fish.

16. the method for claim 11, wherein said Cyprinidae is selected from bright and beautiful carp, zebra fish, Danio or goldfish.

17. the method for claim 11, wherein said Medaka fish are selected from medaka, oviparity Medaka fish or ovoviviparity Medaka fish.

18. the method for claim 11, wherein said zebra fish is selected from D.acrostomus, D.aequipinnatus, D.malabaricus, D.albolineatus, D.annandalei, D.apogon, D.apopyris, D.assamensis, D.choprae, D.chrysotaeniatus, D.dangila, D.devario, D.fangfangae, leopard line zebra, D.fraseri, D.gibber, D.interruptus, D.kakhienensis, D.kyathit, D.laoensis, D.leptos, D.maetaengensis, D.malabaricus, D.naganensis, D.neilgherriensis, D.mgrofasciatus, D.pathirana, D.regina, D.rerio, D.roseus, D.salmonata, D.shanensis, D.spinosus, leopard line zebra or colorful leopard line zebra.

19. the method for claim 17, wherein said Medaka fish is selected from Oryzias javanicus, Oryzias latipes, Oryzias nigrimas, Oryzias luzonensis, Oryzias profundicola, Oryzias matanensis, Oryzias mekongensis, Oryzias minutillus, Oryziasmelastigma, Hong Kong medaka, O.celebensis., X.oophorus, O.celebensis. or X.saracinorum.

20. the process of claim 1 wherein that having genetically modified filial generation is selected from red TK-1x Hong Kong medaka, green TK-1x Hong Kong medaka, red TK-2x leopard line zebra, the colorful leopard line of red TK-2x zebra, green TK-2x leopard line zebra, green TK-2x colorful leopard line zebra or purple fluorescence fairy maiden.

21. the method for claim 20 wherein has genetically modified filial generation and is selected from red TK-1x Hong Kong medaka, green TK-1x Hong Kong medaka, red TK-2x leopard line zebra, red TK-2x colorful leopard line zebra or purple fluorescence fairy maiden.

22. the method for claim 21 wherein has genetically modified filial generation and is selected from red TK-2x leopard line zebra or the colorful leopard line of red TK-2x zebra.

23. a pet fish, its method by claim 1 is produced.

24. the pet fish of claim 23, wherein said pet fish is selected from red TK-1x Hong Kong medaka, green TK-1x Hong Kong medaka, red TK-2x leopard line zebra, the colorful leopard line of red TK-2x zebra, green TK-2x leopard line zebra, green TK-2x colorful leopard line zebra or purple fluorescence fairy maiden.

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