WO2012002610A1 - Double transgenic sterile oryzias dancena - Google Patents

Abstract

The present invention relates to transgenic allotriploid sterile fish that acquire a novel fluorescent phenotype, and more particularly, to double transgenic Oryzias dancena which can express not only strong fluorescent expression in the skeletal muscle but also ubiquitous fluorescent expression in all other tissue, and to sterile allotriploid produced from the double transgenic Oryzias dancena. This novel transgenic allopolyploid can be used as a fluorescent ornamental organism with good ornamental quality because it has a novel fluorescent phenotype which is significantly improved over typical fluorescent transgenic fish; there is no risk of ecosystem transition of foreign genes through genesiological means even through intentional or unintentional release to the environment because of the perfectly sterile characteristic of the fish; and various options can be provided to consumers in the ornamental fish markets because said fish can live and grow normally in fresh water, brackish water, and seawater conditions.

Description

Double Transformation Infertile Fluorescent Sea Killerfish

The present invention provides a transgenic allotriploid infertile fish that obtains a novel fluorescent phenotype through gene transfer, interspecific breeding and chromosome-set manipulation by regulation of novel nucleic acid fragments. More specifically, a double transgenic sea scorpion (Originals dansena) capable of simultaneously expressing strong fluorescence in skeletal muscle and ubiquitous fluorescence in all other tissues. Oryzias dancena ) and sterile allotriploids generated from them.

Fish transformation technology is recognized as an important research technique in the developmental genetics and genomics studies of vertebrates, and also by obtaining new traits (fast growth, disease resistance, nutritional metabolic manipulation, etc.) that are economically important to useful fish species. It is also important as a strategy to drastically improve the productivity of the biological industry (Hackett, PB & Alvarez, MC (2000) .The molecular genetics of transgenic fish.In Recent Advances in Marine Biotechnology , Vol. 4 (Fingerman, M. & Nagabhushanam, R., eds), pp. 77145.Enfield, NH: Science Publishers Inc .; Nam YK, Maclean N, Fu C, Pandian TJ, Eguia MRR (2007) Development of transgenic fish: scientific background.In: Kapuscinski AR , Hayes KR, Li S, Dana G (eds.) Environmental risk assessment for genetically modified organisms.Vol 3. CABI Press, Cambridge, pp 6194; Nam YK, Maclean N, Hwang G, Kim DS (2008) Autotransgenic and allotransgenic manipulation of growth traits in fish for aquaculture: a review.J. Fish Biol. 72: 126; Nam, YK, Noh, JK, Cho, YS, Cho, HJ, Cho, KN, Kim, CG & Kim, DS (2001) .Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis.Transgenic Research 10, 353362). In addition, by implanting living fluorescent protein genes in small fish, it is also used as a new method for producing tubular fish. Transgenic ornamental fish have been developed so far with a fluorescent transgenic strain for zebrafish ( Danio rerio ) and freshwater Japanese medaka ( Oryzias latipes ), and in other small fish species. Experimental studies have been attempted to develop fluorescent fish (Gibbs PD, Schmale MC (2000) GFP as a genetic marker scorable throughout the life cycle of transgenic zebrafish.Mar. Biotechnol. 2: 107125; Chou CY, Horng LS, Tsai HJ (2001) Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation transgenic Res 10:... 303315; Gong Z, Ju B, Wan H (2001) Green fluorescent protein (GFP) transgenic fish and their applications Genetica 111: 213225; Pan X, Zhan H, Gong Z (2008) Ornamental expression of red fluorescent protein in transgenic founders of white skirt tetra (Gymnocorymbus ternetzi) .Mar . Biotechnol. 10: 497501)

However, the commercial use of transgenic fish is recognized for its usefulness in terms of creating new added value in many bioindustry sectors.However, intentional or unintentional release of transgenic fish may lead to biodiversity. Simultaneously contains very negative potential risks (Maclean, N. & Laight, RJ (2000) .Transgenic fish: an evaluation of benefits and risks. Fish and Fisheries 1, 146172; Kapuscinski, AR (2005). the environmental biosafety of transgenic fish and shellfish.Revue Scientifique et Technique de l Office International des Epizooties 24, 309322). In the case of fish, unlike terrestrial genetically modified grains, the mobility is so high that the recovery of unwanted environmental releases of genetically modified fish is not practically possible and the range of risk ecosystems is very wide (Kapuscinski, AR). , Hayes, KR, Li, S. & Dana, G. (2007) .Environmental Risk Assessment of Genetically Modified Organisms, Volume 3: Methodologies for Transgenic Fish.Oxfordshire: CABI Publishing.). Therefore, for the safe commercial use of transgenic fish, reproductive biological sequestration that can prevent the propagation of transgenes in transgenic fish must be accompanied by minimal measures. In particular, for genetically modified fish for ornamental purposes, firstly, it is necessarily distributed in living form to an unspecified number of end consumers, and secondly, frequent exchanges and small transactions among consumers or enthusiasts occur, even after the first consumption phase. Considering that additional distribution of living genetically modified organisms can easily occur, and third, there is a high likelihood of intentional release of living genetically modified fish (eg, indiscriminate release, etc.) due to consumer preference changes. Although fish are generally used indoors, there is always the possibility of causing ecological hazards from environmental releases. Therefore, for the commercialization of transgenic ornamental fish, biological isolation methods that can block the gene transfer of the reproductive pathways that can be caused by environmental release are of great importance.

The case of the development of fluorescent tubular fishes by the conventional transformation technique is mainly zebrafish and Japanese freshwater killari. In the development of fluorescent zebrafish, black halftones and the like exist on the surface of the zebrafish, and albino or golden mutant strains are mainly used to produce highly commercial fluorescent fish. Previously developed fluorescent zebrafish strains include gene regulatory site fragments that can specifically express foreign proteins in skeletal muscle (e.g., myosin light chain 2 polypeptide gene promoter, a-actin gene promoter or muscle creatine kinase gene promoter, etc.). ), It is mainly used in skeletal muscle system, but it shows strong fluorescence expression, but in fins and eyes etc., it is not possible to induce coronary fluorescence phenotypes, and only the muscle expresses fluorescence (Xu Y, He J). , Tian HL, Chan CH, Liao J, Yan T, Lam TJ, Gong Z (1999) Fast Skeletal Muscle-Specific Expression of a Zebrafish Myosin Light Chain 2 Gene and Characterization of Its Promoter by Direct Injection into Skeletal Muscle.DNA AND CELL BIOLOGY 18: 85-95; Ju B, Xu Y, He J, Liao J, Yan T, Hew CL, Lam TJ, Gong Z (1999) Faithful Expression of Green Fluorescent Protein (GFP) in Transgenic Ze brafish Embryos Under Control of Zebrafish Gene Promoters DEVELOPMENTAL GENETICS 25: 158167; Gong Z, Ju B, Wan H (2001) Green fluorescent protein (GFP) transgenic fish and their applications Genetica 111:.. 213225). In some studies, promoter regulation of constitutive and ubiquitous genes that can induce foreign gene expression in various tissues, including beta-actin gene promoters involved in the formation of zebrafiche cytoskeleton (Gibbs PD, Schmale MC (2000) GFP as a genetic marker scorable throughout the life cycle of transgenic zebrafish.Mar. Biotechnol. 2: 107125; Chou CY, Horng LS, Tsai HJ) (2001) Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation.Transgenic Res. 10: 303315; Burket CT, Montgomery JE, Thummel R, Kassen SC, FaFave MC, Langenau DM, Zon LI, Hyde DR ( 2008) Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters.Transgenic Res. 17: 265279). However, transgenic fish prepared by implanting fluorescent expression vectors regulated by these constitutive gene promoters, although capable of inducing a universal fluorescence phenotype in various tissues, have their fluorescence intensity using the muscle specific gene promoter. Since it is much lower than the case, there is a lack of excellent commerciality as a fluorescent tubular fish. In particular, the beta-actin promoter induces high gene expression in many tissues and organs, such as internal organs, but relatively low activity in skeletal muscle, it is likely to be limited in generating a good external fluorescence phenotype. Fluorescent fishes were also developed in the case of Japanese freshwater killifish using beta-actin gene promoter fragments or zebrafish muscle specific expression gene promoters previously used for zebrafish transformation (Hamada K, Tamaki K, Sasado T, Watai Y, Kani S, Wakamatsu Y, Ozato K, Kinoshita M, Kohno R, Takagi S, Kimura M (1998) Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes Mol. Mar. Biol. Biotechnol. 7: 173180; Hsiao CD, Tsai HJ (2003) Transgenic zebrafish with fluorescent germ cell: a useful tool to visualize germ cell proliferation and juvenile hermaphroditism in vivo. Dev. Biol. 262: 313323 ; Zeng Z, Liu, Seebah S , Gong Z (2005) Faithful Expression of Living Color Reporter Genes in Transgenic Medaka Under Two Tissue-Specific Zebrafish Promoters DEVELOPMENTAL DYNAMICS 234: 387392), and they, too, a phenomenon observed in Zebra Fish It shows a similar result. In addition, the conventional fluorescence zebrafish and Japanese killifish are both freshwater fish, and in the seawater environment, normal growth and smooth breeding are not easy. In other words, the use of fluorescent tubular fishes using transformation techniques is limited to freshwater environments.

In view of the above problems, in the present invention, the dissolution of the excellent fluorescence phenotype that is impossible in the conventional single transformation method, ie, strong fluorescence expression in skeletal muscle and excellent coronary value that can simultaneously express fluorescence in all other tissues An attempt was made to provide transgenic tubular fish. In addition, the present invention has been made to provide a safer method of using genetically modified fluorescent fish that is reproductively isolated by inducing infertility through cross-breeding and chromosomal manipulation from transgenic fish.

An object of the present invention is to use two different types of novel regulatory region fragments, a double transformed homozygous sea starfish ( Oryzias dancena ), which simultaneously acquires strong fluorescence expression in skeletal muscle and universal fluorescence expression in all other tissues. Lineages are generated, and from these chromosome manipulations and cross-breeding programs to provide new dissociated fluorescent transgenic ornamental fish that are reproductively isolated.

In order to achieve the above object, in the present invention, the myosin light chain polypeptide isoform 2-2 (mlc2-2) gene promoter and red fluorescent protein (red) of Oryzias dancena fluorescent protein (RFP) structural gene, and further comprising an O. dancena mlc2-2 3'-UTR fragment, preferably comprising a polyadenylation signal, A fluorescence expression vector expressing strong fluorescence in O. dancena muscle; And,

A sea larva comprising a beta-actin gene promoter of the Oryzias dancena and a red fluorescent protein (RFP) structural gene, preferably comprising a polyadenylation signal O. dancena ) provides a fluorescence expression vector that expresses universal fluorescence in the entire O. dancena tissue, further comprising a beta-actin 3'-UTR fragment.

In addition, in the present invention, microtransplanted and incubated the transgenic gene unit in the Oryzias dancena fertilized egg in each of the expression vectors to obtain a transgenic sea trout (F0), which is cross-linked with an untransformed sea trout A method of obtaining a hemizous transformed sea fish (F1) and crossing between transgenic males and females to produce a homozygous transformed sea fish (F2); And

Produced by this method, transgenic marine celery that expresses strong fluorescence in muscle, and transgenic marine celery that expresses universal and always constitutive fluorescence in whole language while expressing relatively weak fluorescence in muscle.

In order to achieve the final object of the present invention, transgenic sea larvae and muscles expressing strong fluorescence in the muscle produced by the method, while expressing relatively weak fluorescence in the muscle and expressing constitutive and constitutive fluorescence in the whole body Intraspecific crosses of transgenic sea scorpions to obtain hemizygous double transgenic sea scorpions and cross-transgenic male and female crosses to produce homozygous double transgenic sea scorpions; And it provides a double transformed sea celery produced by this, which simultaneously express fluorescence in all tissues with strong fluorescence in muscle.

Ultimately, in the present invention, a method for producing an infertile double-transformed marine trellis hybridtriploid comprising the step of interspecific hybridization with the above-mentioned marine trout and Oryzias javanicus , And it is produced by the fluorescence in the whole tissue with the strong fluorescence in the muscle at the same time to provide a fertility double transgenic Oryzias dancena hybrid (lotriploloid) removed fertility.

In the present invention, a novel promoter capable of inducing muscle-specific or constitutive foreign gene expression from Oryzias dancena , which is capable of normal growth in both freshwater, brackish and seawater environments, is fused with fluorescent protein genes. Transformed vectors were prepared. Transgenic homozygous strains expressing strong fluorescence expression in muscles and transgenic homozygous strains capable of expressing universal fluorescence in all tissues through transplantation program after transplanting each transgenic vector to the sea fern through gene transplantation technique A double transformation (simultaneous expression of fluorescence resistant to muscle and universal fluorescence expression in several tissues) with two fluorescence expressing traits was generated by a hybridization program between these two homozygous lines. In addition, for the reproductive isolation of these double transformed strains, a novel transgenic sea with full reproductive capacity with preservative double fluorescence preserved by heterogeneous cross-breeding and induction of ploidy with a related species, Oryzias javanicus . A celtic hybrid triploid could be prepared.

The outline of the configuration of the present invention is as follows.

First, the myosin light chain 2 polypeptide isoform # 2 gene promoter (mlc2-2 promoter), a promoter capable of inducing strong gene expression specifically for skeletal muscle, and relatively low activity in skeletal muscle, A new beta-actin gene promoter, which can induce universal gene expression across all tissues of the whole fish body, is newly isolated from O. dancena and a red fluorescent protein (RFP) structural gene. Construct a fusion gene with Then sea medaka (O. dancena) mlc2-2 gene and beta-by the 3'-UTR (untranslated region) of the actin gene isolated a fragment connected to the respective ends of the two fusion genes, sea medaka (O. dancena) in muscle Two fluorescent expression vectors can be constructed that can express strong fluorescence or can express universal fluorescence in whole tissues.

Second, the transgene unit fragments are separated from the expression vectors, and microinjection is performed in the O. dancena fertilized eggs, respectively, and the transgenic fish selection and breeding programs are used to strengthen the muscles. sea medaka expressing fluorescence (O. dancena) transformed homozygous system and muscle in the relatively weak expression of the fluorescent light at all times, but common and constitutive transformed to express a fluorescent sea medaka (O. dancena) isomorphic in whole body Establish each of the junctional lines.

Third, a double transgenic fluorescence ocean starfish ( O. coli) expressing fluorescence simultaneously in all tissues such as eyes, fins, head, and gills, with strong fluorescence in muscle through intraspecific cross between the two transgenic homozygous lines . dancena ) lines and establish a line that is homozygous for both transgene loci from them through a breeding program and progeny test.

Fourth, interspecific hybridization with O. javanicus , a related species, is carried out for the reproductive isolation of the double transgenic sea larvae ( O. dancena ), at which temperature Perform stimulation treatment. This provides a final product of the present invention by inducing hybrid triploids and verifying the integrity of the double fluorescence phenotype and the suppression of gonad development.

1 is a schematic diagram showing the configuration and flow of the present invention. As shown here, the composition of the present invention allows the use of overlapping transformation, mating and chromosomal manipulation programs to secure new complex traits of fluorescent coronary fish that have not been seen in the prior art, and ecologically through complete reproductive isolation. It provides a safer way to use genetically modified ornamental fish.

Hereinafter, the present invention will be described in more detail.

(1) Preparation of Expression Vector for Fluorescent Transgenic Fish Production

Myosin light chain polypeptide isoform 2-2; mlc2-, responsible for the formation and motor function of skeletal muscle from the photochlorinated sea larvae ( O. dancena ) for the production of transgenic fish. 2) Isolates the gene and its regulatory region, and the beta-actin gene, which is a constitutively expressed gene involved in the cytoskeletal formation, and its regulatory region. Obtaining the cDNA sequences of the mlc2-2 and beta-actin genes respectively from the sea pod expression gene transcripts database constructed for the present invention, from which genomic DNA genes using PCR (polymerase chain reaction) method Obtain fragment sequencing. Genome walking in five directions is performed based on each gene cDNA or gDNA sequence, and 5-upstream regions are secured for each mlc2-2 and beta-actin genes. Using each of these two gene promoters to prepare a fusion structure with a red fluorescent protein (RFP; provided by Clontech's pDsRed2 vector), the obtained sequencing fragments were ligated into pre-prepared pDsRed2 fragments. The ligation) reaction inserts each of the gene promoter fragments immediately before the RFP gene. In addition, the O. dancena mlc2- containing a polyadenylation signal to the NotI position present in pDsRed2 immediately after the RFP gene termination codon, to facilitate the expression of the RFP gene in Origian single cells or cells. 2 3'-UTR fragments or beta-actin 3'-UTR fragments are further inserted respectively. As a result, podmlc2-2RFP as a fluorescent expression vector capable of inducing strong red fluorescence expression in O. dancena muscle and podb as an expression vector capable of expressing universal fluorescence in all O. dancena tissues -finally build actRFP

(2) Transformation Fluorescence Sea Scallop Preparation and Homozygous Line Establishment

To prepare a fluorescent transgenic sea stalk ( O. dancena ), KpnI and AatII restriction enzyme cleavage (for podmlc2-2RFP) or SalI and AatII restriction enzyme cleavage (podb-actRFP) from each of these two plasmid vectors ) Isolates only the transgene units linked to the promoter (mlc2-2 or beta-actin promoter) -RFP gene-3'-UTR (mlc2-2 or beta-actin 3'-UTR) Purify purely. Transgenic gene fragments were micro-injected into single-celled or fertilized Oocytes and screened for the adult embryos that were hatched from the micro-implanted eggs using fluorescence microscopy. do. Select F0 (founder generation) individuals that maintain fluorescence until adulthood, and select transgenic F0 individuals that can deliver fluorescence expression to F1 generation through crossbreeding with offspring and progeny assays. Select again. Phenotypic characteristics expressing strong red fluorescence expression in the skeletal muscle were identified from the mlc2-2-RFP transgenic gene transplant group by selecting fluorescence expressing transgenic F1 individuals, growing to adult and analyzing the fluorescent phenotype. In contrast, the beta-actin-RFP transplantation group identifies phenotypic features that express red fluorescence that is universal in whole language. These two strains were subjected to brother-sister mating of female F1 and male F1 in each transgenic line to generate homozygous F2 individuals, and again to F3 offspring through assay crosses with common sea ferns. Optimal homozygous systems capable of delivering fluorescent traits to all 100% were established in the beta-actin-RFP group and the mlc2-2-RFP group, respectively.

(3) Manufacture of Double Transformed Fluorescent Sea Lobster and Establishment of Homozygous Line

Intracellular hybridization between two established homozygous strains (mlc2-2RFP and b-actRFP strains) (transgenic transbreeding) establishes novel fluorescence expression in all tissues as well as strong fluorescence expression in muscle Produce transgenic individuals of a double fluorescent phenotype. Cross-breeding between transgenic lines is carried out by exchanging male and female between the homozygous lines, thereby generating cross-transgenic hybrid fish that have obtained the desired phenotype. Selected transgenic fishes express strong red fluorescence in skeletal muscle by the mlc2-2 promoter, and double traits in combination with universal red fluorescence in all tissues including eyes, fins, gills and epidermis by the beta-actin promoter. Check the switching system. Strain hybridization is performed to generate individuals that are homozygous for both transgenic loci from the resulting double transgenic individuals. First, the test hybridization confirms that the two transgenes are not linked to each other on the chromosome, and then a sibling cross is performed to select homozygous double transgenic individuals formed by independent genetic methods of the two transgenes. And assay for 100% transfer of double transformation characteristics from selected homozygotes later.

(4) Infertility Hybrid Triploid Double Transform Fluorescent Sea Lobster Preparation

Heterologous hybrid ploidy is induced for reproductive isolation of the newly formed double transgenic fluorescence marine trout lineage. Most of the larvae, including the Occitanus dansena used in the present invention, have very high spawning power and show year-round spawning characteristics, so that when the transgenic ornamental fish using them is intentionally or unintentionally exposed to the environment, allied or inferior to the ecosystem Breeding with species is not only likely to contaminate ecosystem genes, it is also likely to occupy a wide range of ecosystems. Therefore, in order to ensure ecological safety of the double transgenic fluorescent killing individuals produced by the present invention, it is intended to provide infertile transformants in which gonad development and scattering function are suppressed. Currently, mitosis suppression by induction of triploid is widely used as a method of infertility in fish, but in some species, induction of triploid alone, especially in triplode males, does not result in perfect gonad infertility. may have been that the reported (Arai K (2001), Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan Aquaculture 197, 205228;.. Lee CS, Donaldson EM (2001) General discussion on reproductive biotechnology in finfish aquaculture Aquaculture 197 , 303320). In view of this point, the present invention intends to provide a perfect infertile transformant in both male and female by inducing a heterologous hybrid triplet with a related species rather than a simple triplet.

As a candidate species for inducing cross-hybrid drainage , in the present invention, the recently dismissed pine-fish O. javanicus which can maintain the photo-inflammatory characteristics and the transparent body color of O. dancena even in the hybrid drainage was selected. Homozygous double transgenic O. dancena females and normal O. javanicus males are bred and temperature-stimulated to suppress the release of the second polar body of hybrid fertilized eggs. The temperature stimulation treatment is carried out for 30 to 45 minutes at 0 ° C. after fertilization and induces embryonic development and hatching from the fertilized eggs. In the transformed individuals obtained from the hybrid triploid treatment group, hybridoploid formation was confirmed by using DNA content and specific gene fragment sequencing method, and the identified individuals were grown to express double traits and have a reproductive isolation. Check.

The heterologous hybrid triploid double fluorescent transgenic fish generated according to the present invention stably well preserves the double transformation characteristics (ie, strong fluorescence of muscle and universal fluorescence expression in all other tissues) of the mother O. dancena . can confirm. In addition, transgenic triploids have not been shown to produce viable fertilized eggs at all in long-term induction with normal O. dancena or O. javanicus , so that these hybrid heteroploid transgenic sea larvae are functionally sterile in both male and female. Proved to be obtained.

1 is a schematic view showing the configuration and flow of the present invention,

2 is a schematic diagram of the podmlc2-2RFP fluorescence expression vector,

3 is a graph showing the expression distribution and level of mlc2-2 mRNA in adult tissues of Oryzias dancena ,

4 is a schematic diagram of the podb-actRFP fluorescence expression vector,

5 is a graph showing the tissue expression distribution and level of beta-actin mRNA in adult Oryzias dancena tissue,

Figure 6 is a photograph showing the fluorescence expression in the Oryzias dancena embryo (embryo) micro-injected with the transgene mlc2-2-RFP,

7 is a photograph showing the appearance of the F1 transgenic sea larvae ( Oryzias dancena ) and general sea larvae ( Oryzias dancena ) to which the transgene mlc2-2-RFP is transplanted.

Figure 8 is a photograph showing the fluorescence expression in the Oryzias dancena embryo (embryo) micro-injected with the transgene beta-actin-RFP,

FIG. 9 is a photograph showing RFP expression in F1 transgenic Oryzias dancena appearance and external tissues transfected with transgene beta-actin-RFP.

10 is a photograph showing RFP expression in various internal organs of the F1 transgenic sea trout Oryzias dancena transplanted with the transgene beta-actin-RFP,

FIG. 11 is a double transformed Oryzias dancena photograph expressing fluorescence in whole tissues with strong fluorescence in muscle.

12 shows PCR amplification of the transgenes from the double transgenic sea larvae ( Oryzias dancena ),

FIG. 13 is a photograph of an infertile transgenic hybrid triploid generated through cross-breeding and induction of ploidy with a double transgenic sea trout ( Oyzias dancena ) and O. javanicus .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are intended to more specifically illustrate the configuration and effects of the present invention, the scope of the present invention is not limited to these.

Example 1 Preparation of Fluorescence Expression Vector Comprising the Sea Squid mlc2-2 Promoter

EST (expressed sequence tag) analysis was performed from cDNA library constructed using total RNA of O. dancena whole body, and mlc2-2 cDNA sequence (SEQ ID NO: 1) was obtained through EST database analysis. Based on the obtained mlc2-2 cDNA gene fragment sequence, genome walking was performed to secure a regulatory region including a promoter of the upstream mlc2-2 gene. Genome walking was performed according to the manufacturer's recommended method using the Universal Genome Walker kit (Clontech Laboratories, USA), wherein two reverse primers specific to the O. dancena mlc2-2 gene (ODMlc2-2 GW1; No. 2 and ODMlc2-2 GW2; SEQ ID NO: 3) were synthesized and used. The reaction composition and PCR amplification conditions for genome walking are shown in Table 1 below. Second genome walking was carried out in the same manner based on the results of the first genome walking, in which two reverse primers (ODMlc2-2 GW3; SEQ ID NO: 4 and ODMlc2-2 GW4; SEQ ID NO: 5) were used. Table 1 below shows PCR reaction compositions and amplification conditions for genome walking to the MLC2-2 promoter region.

Table 1

Using a 5-upstream regulatory region fragment (SEQ ID NO: 6) of the mlc2-2 gene obtained through genome walking, a fusion structure with a red fluorescent protein (RFP; provided by Clontech's pDsRed2 vector) gene was prepared. To this end, the obtained sequence fragments were separated again by PCR. Table 2 shows reaction compositions and amplification conditions for PCR separation of the mlc2-2 promoter. In this case, KpnI at each 5-terminal of the PCR primer (forward; KpnI-ODMlc2-2 1F; SEQ ID NO: 7) and reverse (AgeI-ODMlc2-2 1R; SEQ ID NO: 8) to facilitate the fusion with the RFP gene. A restriction enzyme recognition sequence (AAT GGTACC ) and a restriction enzyme AgeI recognition sequence (AAT ACCGGT ) were artificially inserted, respectively. The PCR product was cloned into the pGEM T-easy vector (Promega) according to the manufacturer's recommended method, and the fragments were recovered by KpnI and AgeI restriction enzyme treatment, and then ligation with the pDsRed2 fragment prepared in advance through the same restriction enzyme treatment. Gene promoter fragments were inserted immediately before the RFP gene, respectively. Table 3 shows the ligation reaction compositions and reaction conditions for the fusion structure of the mlc2-2 promoter with the RFP gene.

TABLE 2

TABLE 3

Upon completion of the ligation reaction, 5 μl of the reaction was used to transduce the E. coli XL-blue MRF ′ strain (Stratagene, USA) according to the manufacturer's recommended method, and recombinant strains were selected to recover the plasmid DNA. Clones with accurate ligation reactions were assayed and selected through sequencing.

For the selected plasmid vector, a polyadenylation signal is sent to the NotI restriction enzyme position immediately after the RFP gene termination codon for easier expression of the RFP gene in O. dancena cells. A sea larvae ( O. dancena ) containing the mlc2-2 gene 3'-UTR fragment (182 bp; SEQ ID NO: 9) was further inserted. The 3'-UTR fragment of the corresponding mlc2-2 gene was subjected to PCR isolation using two primers (forward primer NotI-ODMlc2-2 3UFW1; SEQ ID NO: 10 and reverse primer NotI-AatII-ODMlc2-2 3URV1; SEQ ID NO: 11). Secured through. Table 4 below shows reaction compositions and amplification conditions for PCR isolation of the mlc2-2 gene 3′-UTR region. At this time, the NotI recognition sequence (ATA GCGGCCGC ) was artificially inserted at the two primer 5-terminals for insertion of the pDsRed2 plasmid into the NotI position, and the reverse primers were used to recover only the transgenic gene fragment for later micromicroinjection . An AatII restriction enzyme recognition sequence (ATA GCGGCCGC GACGTC ) was further inserted. As a result, podmlc2-2RFP was prepared as a fluorescence expression vector capable of inducing strong red fluorescence expression in O. dancena muscle. 2 is a schematic diagram of the podmlc2-2RFP fluorescent expression vector.

Table 4

Example 2: Tissue Expression Characterization of Sea Maldid endogenous mlc2-2 Gene

In order to confirm the tissue specific activity of the O. dancena mlc2-2 promoter of the present invention, real-time reverse transcription PCR ( MLC2-2 ) gene mRNA levels were measured in various tissues of the O. dancena ( O. dancena ). Reverse transcription-PCR) method was used to investigate. Various RNAs (brain, gill, heart, digestive tract, kidney, liver, muscle, spleen, gonad) were extracted from each of the 12 male and female marine species and total RNA was extracted using RNeasy Mini-kit (Qiagen, Germany). Separated. CDNA was synthesized according to the manufacturer's recommended method using Omniscript Reverse Transcription System (Qiagen, Germany) on 2 μg of total RNA separated by each tissue, and 18S rRNA was prepared together in the reverse transcription reaction as a normalization control. PCR primer pairs capable of amplifying mlc2-2 mRNA fragments were used for investigating the presence of mlc2-2 mRNA expression and expression levels by sea fern tissues, as follows: forward primer qODMlc2-2 1F (SEQ ID NO: 12) and reverse primer qODMlc2-2 1R (SEQ ID NO: 13) was synthesized, and changes in the amount of real-time PCR amplification were tracked using an iCycler real-time PCR optic module manufactured by Bio-Rad (USA). Table 5 shows PCR reaction compositions and thermocycle amplification conditions for real-time quantitative amplification of mlc2-2 mRNA. The expression level of mlc2-2 mRNA in each tissue was corrected based on the level of the 18S rRNA control group of the tissue, and the relative quantification of the tissues was performed by Kubista et al. (Kubista M, Andrade JM, Bengtsson M, Forootan A, JonJ, Lind). K, Sindelka R, SjR, SjB, StrL, StA, Zoric N (2006) The real-time polymerase chain reaction.Mol Aspects Med 27 : 95125).

Table 5

Figure 3 is a graph showing the expression distribution and level of mlc2-2 mRNA in adult tissues of Oryzias dancena . As shown in FIG. 3, most of the marine trout mlc2-2 gene expression was strongly concentrated in the muscle, and the gene expression was not detected or very poor in the rest of the examined tissues. Therefore, in the present invention, when the fluorescence expression by the mlc2-2 promoter is expressed in the transgenic sea larvae, the fluorescence expression can be strongly concentrated in the muscle.

Example 3: Preparation of Fluorescence Expression Vector Comprising Sea Species Beta-Actin Promoter

The beta-actin promoter was isolated to produce expression vectors capable of inducing universal and always constitutive fluorescence expression in whole sea pods. The entire beta-actin cDNA sequence (SEQ ID NO: 14) was obtained from the sea lychee EST database described in Example 1, and the 5-upstream regulatory region fragment was isolated through genome walking and isolation of genomic gene fragments as in Example 1. Secured. The primers used for isolation and genome walking of genomic beta-actin gene fragments are shown in SEQ ID NOs: 15 to 18. Table 6 shows PCR reaction compositions and amplification conditions for beta-actin genomic gene isolation and genome walking to the promoter region.

Table 6

The beta-actin regulatory region obtained through genome walking is a total of 3.88 kb sequence linked to the 5-upstream region, non-translated exon I and intron I (SEQ ID NO: 19). ). Embodiment similar to that in the first to artificially insert a restriction enzyme SalI recognition sequences (AT GTCGAC) a forward primer, including (ODb-ACTp 2F; SEQ ID NO: 20) and AgeI recognition sequences (AT ACCGGT) reverse primer (ODb-ACTp including PCR was isolated using 2R; SEQ ID NO: 21) and cloned into the RFP gene upstream of the pDsRed2 vector. In addition, in order to facilitate the expression of the RFP gene, as in Example 1, the forward primer containing the NotI recognition sequence (NotI-ODb-ACT 3UFW1; SEQ ID NO: 22) and the reverse primer containing the NotI / AatII recognition sequence (NotI-AatII Beta-actin 3′-UTR fragment (650 bp; SEQ ID NO: 24) was further linked to the RFP stop codon downstream using -ODb-ACT 3URV1; SEQ ID NO: 23) after PCR separation. As a result, podb-actRFP was prepared as a fluorescence expression vector capable of inducing constitutive red fluorescence expression in the whole fish of O. dancena . 4 is a schematic diagram of a podb-actRFP fluorescent expression vector.

Example 4: Tissue Expression Characterization of Sea Trout Endogenous Beta-Actin Gene

In order to confirm whether the sea-fish beta-actin gene expression regulation is universal in all tissues, real-time reverse transcription PCR analysis was performed on adult tissue-specific cDNA prepared in Example 2 above. The composition and conditions of the reactants used were the same as in Example 2, except that the forward primer qODb-ACT 1F (SEQ ID NO: 25) and the reverse primer qODb-ACT 1R (SEQ ID NO: 26) as primer pairs for amplifying beta-actin mRNA fragments. ) Was used. Correction of tissue expression and relative quantification between tissues were also performed in the same manner as in Example 2.

5 is a graph showing the tissue expression distribution and level of beta-actin mRNA in adult Oryzias dancena tissues. As shown here, the sea larvae beta-actin gene shows mRNA expression in all tissues analyzed, demonstrating the universal regulatory characteristics of the beta-actin gene. On the other hand, according to Figure 5, the expression level of beta-actin mRNA is significantly different from tissue to tissue, showing a high expression level in the digestive tract, kidney, spleen and ovary while relatively mild expression in the brain, gills, heart, testis tissue. And the lowest expression in muscle and liver tissues. Therefore, when inducing foreign fluorescence gene expression using sea urchin beta-actin regulatory region, transgenic sea urchin adults can express fluorescence universally in all tissues, but the difference in expression rate is expected in each tissue, especially in the skeletal muscular system. Expression of the gene protein was difficult to expect.

Example 5: Establishment of Fluorescent Sea Killer Line System by Regulating mlc2-2 Promoter

In order to prepare a transgenic sea trout capable of inducing the expression of a strong fluorescent protein concentrated in muscle, the transgene fragments recovered from the podmlc2-2RFP through KpnI / AatII restriction enzyme treatment were microscopically injected into the sea trout fertilized eggs. . To this end, male and male adult males and females were housed in a 30 L tank at 25 ° C. in a male to female ratio of 2: 1 and observed mating and spawning behavior. When spawning was confirmed, the fertilized eggs were recovered immediately, and the fertilized eggs in the first warming season were selected and microscopically injected into the cytoplasm under a microscope. Micromicroscopic injection was performed using a micromanipulator (Narishige, Japan), and the DNA for micromicroscopic injection was adjusted to a concentration of 100 ㎍ / ㎖ in 10 mM Tris-Cl, 0.1 mM EDTA pH 8.0 buffer solution. The presence and pattern of RFP expression in each stage were examined by fluorescence microscopy in 110 first microembryled embryos. As a result, RFP expression by regulation of the mlc2-2 promoter was found to be in good agreement with the characteristics of the mlc2-2 gene regulation as observed in muscle-specific patterns in the late embryo. Figure 6 is a photograph showing the fluorescence expression in the Oryzias dancena embryo (embryo) micro-injected with the transgene mlc2-2-RFP.

In order to produce transgenic fish based on the first analysis result, micro-micron injection was carried out to 981 fertilized eggs again. Of these, 342 individuals (34.9%) hatched and 267 of the hatched individuals reached the final maturation stage. Red fluorescence expression was observed in 35 (13.2%) of 267 adults, and RFP fluorescence expression was all localized to skeletal muscle, demonstrating well-characterized muscle expression by regulation of the mlc2-2 promoter. The generated F0 individuals showed mosaic-like expression, but the F1 transformed individuals generated through crosses with general sea celtices showed very strong fluorescence expression in all skeletal muscles, unlike the F0 parents. Table 7 below shows fluorescence transfer from mlc2-2-RFP transformed F0 to F1 progeny.

TABLE 7

Figure 7 is a picture of a gene transformed appearance mlc2-2-RFP the F1 transgenic medaka sea transplantation (Oryzias dancena) and normal sea medaka (Oryzias dancena). As shown here, the strong red fluorescence can be easily observed with the naked eye without fluorescent lighting, and the fluorescence expression is specific to skeletal muscle, so RFP is applied to the head, upper fin, and upper upper head. It can be seen that there is no expression.

In order to establish a homozygous transformation lineage from F1 hemizygous transgenic individuals, sibling crosses were conducted at a 1: 1 ratio between F1 females and males. Among the F2 transformed phenotype-bearing individuals obtained through sibling breeding, six randomly selected individuals showing relatively strong fluorescence intensity were randomly selected and subjected to assay crosses with general sea scorpions. By investigating the presence or absence of fluorescence expression of the produced F3 progeny, four transgenic individuals capable of delivering 100% fluorescence expression later were established, thereby establishing a line homozygous for the transgenic locus inserted into the chromosome. Table 8 below shows the selection of mlc2-2 homozygous transform F2 through progeny assay.

Table 8

Example 6: Establishment of Fluorescent Sea Glory Lines by Modulation of Beta-Actin Promoter

In order to prepare the original Oris homozygous system for fluorescence expression in whole tissues, SalI / AatII fragments of the transformed expression vector podb-actRFP developed above were microscopically injected. To examine whether the beta-actin promoter of the present invention can induce universal RFP expression in O. dancena cells as a preliminary evaluation experiment, the embryos developed after micro-micro injection of the transgene into fertilized eggs ( Fluorescence was examined in embryos. Micro-injected embryos began to show strong fluorescence expression from the early stages, and the expression patterns of RFP-expressing embryos varied, and in most all cells without cell-specific or developmental-specific patterns. Universal expression characteristics. Figure 8 is a photograph showing the fluorescence expression in the Oryzias dancena embryo (embryo) micro-injected with the transgene beta-actin-RFP.

On the basis of this result, in order to manufacture a full-scale transgenic fish, a total of 1189 fertilized eggs were re-injected with micro-microns, and 386 (32.5%) of them hatched and reached the adulthood where the final individuals could mature. As a result of investigating the external fluorescence phenotype in adults, RFP expression was detected in 28 (10.3%) of the 271 individuals analyzed, 9 of which were sufficient to detect RFP easily with the naked eye without the need for fluorescent illumination. Red fluorescence was shown. Most of the F0 generation showed mosaic-shaped fluorescence distribution, but the expression of the red fluorescence showed fluorescence expression over various tissue types such as gills, fins, and muscles without specific features limited to specific cell or tissue types. Randomly selected of these fluorescence expressing F0 individuals were selected to be capable of delivering transgenic expression to the posterior F1 by mating with a general sea fern. As a result of crossing the control group with a total of 9 F0 individuals finally selected, 5 F0 individuals successfully delivered fluorescence to F1, and the frequency of delivery varied by individual. Table 9 below shows fluorescence delivery from beta-actin RFP transgenic F0 to F1 progeny.

Table 9

In contrast to the F0 generation, where fluorescence expression is observed in mosaic form, all cells show successful expression of RFP fluorescence in all cells and tissues of F1 individuals that contain the transgene in the form of a hemizygote. The effect of the present invention is well illustrated. FIG. 9 is a photograph showing RFP expression in F1 transgenic Oryzias dancena appearance and external tissues transfected with transgene beta-actin-RFP. FIG. These F1 individuals have mild RFP fluorescence intensities throughout the muscle and other tissues show varying intensity of fluorescence per tissue. In the external phenotype, these transgenic individuals express RFP fluorescence in the eyes, scales, head, and fins as well as beta-actin gene expression such as varying fluorescence intensity in various organs such as kidney, gut, spleen, etc. It appeared to follow the characteristics of the well. FIG. 10 is a photograph showing RFP expression in various internal organs of an F1 transgenic Oryzias dancena transplanted with transgene beta-actin-RFP. Thus, the present transgenic lines do not express strong fluorescence in muscle as in the muscle concentrated fluorescence expression line of Example 5, but the universal fluorescence expression distribution in all tissues that cannot be obtained in mlc2-2-RFP transformation Express

F2 transgenic individuals were generated through sibling crosses between female and male F1 individuals of the lineage selected from them. No significant differences in fertilization rate, hatching rate and initial survival rate were found between the F1 transgenic organisms and the normal control, and about 75% of the transgenic genes were found to be consistent with the Mendelian method. Is showing. In addition, it shows that a stable reproductive line was formed in the transgenic transgenic process such as meiosis without reducing or losing transgene expression. F2 progeny of relatively high fluorescence intensities were selected by visual evaluation of fluorescence on the generated F2 progeny, and crossed with general sea scorpions. Subjects were selected. Table 10 below shows the selection of beta-actin RFP homozygous transform F2 through progeny assay.

Table 10

Example 7: Double Transform Fluorescence Sea Species O dancena System establishment

The transgenic lines were transfected with the transgenic homozygotes F2 obtained in Examples 5 and 6. The mating was carried out in a cancer: number = 1: 1 manner, and fertilization rate, hatching rate and initial survival rate of fertilized eggs obtained through laying and mating were investigated. As a result, both fertilization rate and hatching rate were not significantly different from those of the general control group. Therefore, it was judged that double transgenic individuals which simultaneously acquired two transgenic phenotypes could be formed without any obstacle to viability.

As a result of analyzing the fluorescence expression of the transgenic adult, double transgenic individuals expressed all of the parent's fluorescent traits intact, thereby indicating fluorescence expression in all tissues by beta-actin promoter regulation, as well as mlc2-2. Promoter regulation showed strong fluorescence expression in skeletal muscular system. Thus, while addressing the shortcomings of transgenic fluorescence expression by beta-actin promoter alone regulation in skeletal muscle, constitutive fluorescence expression in dorsal, eye and head, which was not obtainable by regulation of the mlc2-2 promoter alone. By acquiring together, it is well shown that a novel fluorescence phenotype that could not be obtained in the past was secured. FIG. 11 is a picture of a double transgenic sea killifish ( Oryzias dancena ) expressing fluorescence in all tissues with strong fluorescence in muscle. FIG.

Double transgenic individuals formed through the mating of two homozygous transgenic parents exhibit a hemizygous state for each transgenic locus, and in the present invention, the two traits are independently transferred when assayed. In the selected strains, the two transgenic loci were not physically related. Accordingly, in order to convert both transgenic loci of the double transformed individuals into homozygous state, sibling crosses were performed again between the double transformed individuals, and double transformation (strong muscle expression and other The incidence of constitutive expression in tissues was about 52%, which was not significantly different from that of the Mendelian 9: 7 (3: 3: 1) mode. A total of 25 of these double-transformed phenotypes were bred with common sea scorpions, and three of them (3/25 = 12%) were homozygous for stably delivering double transformation to the next generation. It appears to be a conjugate, showing that it also follows Mendel's independent inheritance (1: 2: 2: 4) mode. Table 11 shows the selection of double transgenic homozygous transgenic lines through progeny assay.

Table 11

PCR of the mlc2-2-RFP transgene and the beta-actin-RFP transgene to detect the presence of the actual two transgenes in one individual of the selected double transgenic lines. It was. A first forward primer specific for the mlc2-2 promoter (TGODMlc2-2 2F; SEQ ID NO: 27), a second specific to the beta-actin promoter, based on 100 ng of genomic DNA extracted from transgenic fish for PCR detection A multiplex PCR was performed using a forward primer (TGODb-act 1F; SEQ ID NO: 28) and a reverse primer (TGRFP 1R; SEQ ID NO: 29) specific to RFP, a common structural gene of two transgenes. It was. Table 12 below shows the composition of the reactants and the thermal cycling amplification conditions for PCR identification of transformed individuals.

Table 12

Figure 12 shows the results of PCR amplification of the transgenes from the double transgenic sea killi ( Oryzias dancena ). Here, two transgene fragments were detected in all the analyzed individuals, thus showing that the fluorescent phenotype observed in the double transgenic lineage of the present invention was obtained by the operation of the transgene.

Example 8 Preparation of Transgenic Infertility Hybrid Drain Fluorescent Fishes by Induction of Cross-Team Hybrid Drainage

For reproductive isolation of the double transformed homozygotes selected in Example 7, a hybrid diploid double transformant was generated by cross-breeding with oryzias javanicus and inducing diploids. Two females of double transgenic O. dancena and one female of normal O. javanicus were housed in each of the three tanks and hybrid hybridization and spawning were induced. As a scattering tank for the breeding, a 30 L 25 ° C water bath was used, the photoperiod was adjusted to the contrast of 16h: 8h, and the salinity was 15 ppt. Immediately after spawning, fertilized eggs were recovered and the second polar body was suppressed through the temperature stimulation treatment. Temperature stimulation treatment was carried out for 45 minutes at 0 ℃ after 3 minutes of fertilization, and after the treatment was completed and transferred to a 25 ℃ constant temperature incubator (15 ppt) to induce hatching. A total of three iterations were performed. The hybridization rate between these two species was 90.5%, which was not significantly different from that of intrabreeding. The hatching rate was 80.5%, which was slightly lower than that of parental breeding. The average incubation rate of the inter-drainage drainage group treated with temperature treatment was 68.8%, which was significantly higher than that of the untreated diploid hybrid group. The fluorescence microscopy of hybrid embryogenic embryos or embryonic embryos showed 100% RFP fluorescence in all observed samples, so that the transgenes transferred from the female double transgenic O. dancena used were heterozygous for karyogamy. It showed that it can work reliably in the state. Table 13 below shows the production of heterologous hybrids and hybrid triplets by Oryzias dancena (F) × O. Javanicus (M) crosses.

Table 13

In order to confirm the triploid ploidy by suppressing the release of the second polar body by the temperature treatment, the DNA content was examined by flow cytometry in the hybrid triploid transformed individuals. As a result, the average DNA content per cell of the diploid O. dancena female × O. javanicus male hybrid was 1.7 pg / cell, whereas the hybrid triploid was 2.6 pg / cell, which is very similar to the 1.5-fold increase expected. Table 14 shows DNA contents per cell of Oryzias dancena (F) × O. Javanicus (M) hybrids and hybrid triploids.

Table 14

Thus, hybrid diploid double transgenic individuals formed through inhibition of dipolar release are triploid hybrid fish, including two haploid pairs of the maternal (double-transformed O. dancena females) and one paternal (generic O. javanicus male) haploids, The action of the double transgenes derived from the maternal system simultaneously shows strong fluorescence expression in the muscle (mlc2-2 promoter regulation) and fluorescence expression in other tissues (beta-actin promoter regulation), the phenotypic characteristic of which is diploid It was very similar to the phenotype of the maternal line that provided chromosome formation. FIG. 13 is a photograph of an infertile transgenic hybrid triploid generated through cross-breeding and induction of a ploidy between a double transgenic sea larva ( Oyzias dancena ) and O. javanicus .

In order to observe the gonad development of these hybrid ploid double transgenic fishes, we performed an anatomical external examination of the gonad tissues of adult females and males grown for 5 months. As a result, normal O. dancena and O. javanicus adults in all individuals have well-developed ovary and testis tissue, including mature spouses, while hybrid ploids have completely suppressed gonad development, resulting in the observation of extremely small embryonic gonads, or Development was inhibited to the extent that the gonads could not be easily distinguished with the naked eye.

In this way, we investigated whether hybrid ploidy individuals with an undeveloped gonad could form viable fertilized eggs by crossing with common O. dancena and O. javanicus . Table 1 shows the results of egg production and fertilization of eggs by 1: 1 breeding (hybrid ploidy × 6 O. dancena hybrids and 6 hybrid ploidy × O. javanicus hybridization). Table 15 shows the results of evaluating the fertility of the double transformed hybrid triploid.

Table 15

Here, in O. dancena or O. javanicus mating, fertilized eggs were produced through very active mating and spawning for one month, while mating with hybrid drainage did not produce viable fertilized eggs at all. Some O. dancena or O. javanicus females housed with hybrids in some hybrids showed some spawning, but all eggs laid were 100% unfertilized.

As can be seen from the above results, the double transgenic hybrid ploidy individuals produced by the present invention maintained the desired fluorescence phenotype well, but the fertility production was completely eliminated through fertilization with common killing species as a complete functional infertility. Able to know. Thus, the newly formed double transgenic hybrid drainage sea scorpion through the present invention provides a way to distribute and use fluorescent tubular fish in an ecologically safer manner.

The novel transgenic hybrid drainage according to the present invention possesses a novel fluorescent phenotype significantly improved compared to conventional fluorescent transgenic fishes, and thus can be used as a fluorescent coronary organism with excellent coronary value, as well as having perfect sterility characteristics. As a result, there is no risk of ecological transfer of foreign genes through the reproductive pathway even in intentional or unintentional release of the environment, thus enabling the use of safe genetically modified fluorescent tubular fish. In addition, according to the present invention, since it provides a photo-inflammatory transgenic tubular fish capable of normal growth in freshwater, brackish water and seawater conditions, it is possible to provide consumers with a wider choice in the tubular fish market.

Claims (13)

1. Myosin light chain polypeptide isoform 2-2 (mlc2-2) gene promoter and red fluorescent protein (RFP) structural gene in Oryzias dancena Fluorescence expression vector expressing strong fluorescence in O. dancena muscle.
2. The podmlc2-2RFP of claim 1, which is a fluorescent expression vector further comprising an O. dancena mlc2-2 3′-UTR fragment comprising a polyadenylation signal.
3. Fluorescence expressing universal fluorescence in whole O. dancena tissue, including the beta-actin gene promoter of the Oryzias dancena and the red fluorescent protein (RFP) structural gene Expression vector.
4. 4. The podb-actRFP of claim 3, wherein the podb-actRFP is a fluorescent expression vector further comprising an O. dancena beta-actin 3'-UTR fragment comprising a polyadenylation signal.
5. In the vector of claim 1, the transgenic gene unit is micro-injected and incubated in an Oryzias dancena fertilized egg and incubated to obtain a transgenic sea trout (F0), which is then crossed with an untransformed sea trout to form a hemizygous transformation. A method of obtaining a sea trout (F1) and crossing it between transgenic males and females to produce a homozygous transformed sea trout (F2).
6. A transformed sea trout ( Oryzias dancena ) produced by the method of claim 5 which expresses strong fluorescence in muscle.
7. In the vector of claim 3, the transgenic gene unit is injected microscopically into an Oryzias dancena fertilized egg and incubated to obtain a transgenic sea trout (F0), which is cross-bred with an untransformed sea trout to form a hemiygous transformation. A method of obtaining a sea trout (F1) and crossing it between transgenic males and females to produce a homozygous transformed sea trout (F2).
8. Produced by the method of claim 7, transgenic sea larvae ( Oryzias dancena ), which expresses relatively weak fluorescence in muscle and expresses universal and constitutive fluorescence in whole language.
9. Intraspecific crosses of the Oryzias dancena of claim 6 and the marine scorpion of claim 8 to obtain hemizygous double transformed sea larvae, and crossover between double transgenic males and females. How to produce a conversion sea scorpion.
10. A double-transformed sea larva ( Oryzias dancena ) produced by the method of claim 9, which simultaneously expresses fluorescence in all tissues with strong fluorescence in muscle.
11. A method of producing an infertile double transgenic marine trellis (allotriploid) comprising the step of interspecific hybridization of an Oryzias dancena of claim 10 with an Oryzias javanicus .
12. The method of claim 11, further comprising the step of treating the fertilized egg at 0 ° C. for 30 to 45 minutes to inhibit release of the second polar body.
13. An infertile double transgenic Oryzias dancena hybridtriploid , produced by the method of claim 11 or 12, which simultaneously expresses fluorescence in all tissues with strong fluorescence in muscle and has been removed from fertility.

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