TWI464264B - Production method of transgenic goldfish and gene expression vector for the same - Google Patents

Description

Method for producing gene-transferred goldfish and gene expression vector used in the method

The present invention relates to a method for producing a genetically-transferred goldfish and a gene-transferred goldfish obtained by the method.

In recent years, a variety of genes have been introduced into zebrafish (Zebrafish) and the like to produce fish that express gene transfer (see, for example, Patent Document 1). These gene-transformed fishes are used as model animals for investigating the effects of the introduced genes on fish, or animals for producing useful proteins.

In the manufacture of these genetically modified fish, zebrafish has been mainly used as a target fish. The zebrafish has a small body shape of about 4 cm and is easy to raise, and has many advantages such as easy egg collection. On the other hand, since the body size is small and it is difficult to collect blood or the like, it is difficult to obtain a problem such as serum even if an antibody is produced.

Here, the inventors of the present invention have focused on goldfish as a fish that can replace zebrafish. Goldfish is larger than zebrafish, and blood collection is easier.

The use of goldfish as one of the target fish for gene transfer has not been suggested (for example, refer to Patent Document 1), and is not specifically described, and it is expected to provide a specific method for producing a genetically-transferred goldfish and by the method. The gene is transferred to goldfish.

[Previous Technical Literature] [Patent Literature]

Patent Document 1: Japanese Patent Publication No. 2005-511052

The subject of the present invention is to provide a method for producing a genetically-transferred goldfish and a gene-transferred goldfish obtained by the method.

As a result of intensive research, the present inventors have found that when a gene expression vector incorporating a target gene is introduced into a goldfish, a gene-transferred goldfish expressing a target gene can be produced, and the present invention has been completed.

That is, the present invention relates to a method for producing a gene-transferred goldfish, a gene-transferred goldfish, and the like according to the following (1) to (11).

(1) A method for producing a genetically-transferred goldfish, which comprises the step of introducing a gene expression vector into which a target gene is introduced into a fertilized egg of a goldfish.

(2) The method for producing a genetically-transferred goldfish according to (1), wherein the gene expression vector contains pXI, pZex or pZEF1 as a promoter.

(3) The method for producing a gene-transferred goldfish according to the above aspect, wherein the gene expression vector contains at least two of pXI, pZex and pZEF1 as promoters.

(4) The method for producing a genetically-transferred goldfish according to (3), wherein two or more of pXI, pZex, and pZEF1 are arranged in series.

(5) The method for producing a genetically-transferred goldfish according to any one of (1) to (4) wherein the target gene system is expressed throughout the body.

(6) The method for producing a genetically-transferred goldfish according to any one of (1) to (5), wherein the goldfish is selected from the group consisting of gold, gold, Zhu Wenjin, Huixing goldfish, Tosajin, Dijin, and Chaotianyan. , Hydrangea, Pearl Scale, Osaka Lanshou, Butterflytail, Panda Goldfish, Sakura Jin, Dragon Eye, Wuhua Jinjin, Lanshou, Dutch Lion Head, Dongjin, Edo Jin, Binjin, Danding, Qingwen Fish, Zi Gao Head and any of Nanjing.

(7) A gene expression vector which is a gene expression vector used in a method for producing a gene-transferred goldfish, which comprises pXI, pZex or pZEF1 as a promoter.

(8) The gene expression vector according to (7), which contains at least two of pXI, pZex and pZEF1 as promoters.

(9) The gene expression vector according to (8), wherein any two or more of pXI, pZex, and pZEF1 are arranged in series.

(10) The gene expression vector according to any one of (7) to (9), which is introduced into a fertilized egg of a goldfish.

(11) The gene expression vector according to any one of (7) to (10), wherein the goldfish is selected from the group consisting of gold, gold, Zhu Wenjin, Huixing goldfish, Tosajin, Dijin, Chaotianyan, Hydrangea, Pearl Scale, Osaka Lanshou, Butterflytail, Panda Goldfish, Sakurajin, Longjing, Wuhuajinjin, Lanshou, Dutch Lion Head, Dongjin, Edojin, Binjin, Danding, Qingwenyu, Zi Gaotou and Nanjing By.

According to the present invention, gene transfer goldfish can be produced. The gene-transferred goldfish of the present invention is used as a model animal for investigating the influence of the introduced gene on fish, or an animal for producing a useful protein.

[To implement the type of invention]

The "gene-transferred goldfish" of the present invention may comprise any type of goldfish if it is a gene-transferred goldfish obtained by introducing a gene to be introduced. A gene can be transferred to a goldfish for a gene expressed in a part of the body, or a gene that can be expressed in the whole body of a target gene.

Here, the "target gene" refers to a gene for expression purposes. For example, when a specific gene is introduced, the specific gene is a "target gene" for the purpose of investigating the influence of the gene. Further, when a specific protein is produced, the gene encoding the protein becomes a "target gene." Specific examples thereof include exogenous genes derived from goldfish, and the like, and these genes may be combined with genes encoding fluorescent substances such as EGFP and DsRed for confirming the expression of genes as "target genes." "."

The "gene-transferred goldfish" of the present invention can be produced by introducing a gene expression vector into which a target gene is introduced into a fertilized egg of a goldfish, an unfertilized egg, or an adult body cell.

The "gene expression vector" to which the target gene is incorporated may be any one that can express the target gene, for example, pXI containing the Xenopus laevis elongation factor 1-alpha promoter, and zebrafish hatching-grand-enzyme The pZex of the promoter (both in reference) 1 and the pZEF1 of the zebrafish elongation factor 1-alpha promoter exclusively produced by the present inventors as a gene expression vector of a promoter.

The "gene-transferred goldfish" of the present invention may contain one or more promoters for expressing a target gene, and may also contain a plurality of promoters. Moreover, as a promoter capable of expressing a gene of interest, it may be part of the promoter. For example, a part of pZex shown by the sequence number SEQ ID NO: 4 may be mentioned. In the present invention, a part of the promoters may also be referred to as a "promoter".

When a plurality of promoters are included, the promoter may be arbitrarily arranged, for example, a gene expression vector in which promoters are arranged in series may be used.

Reference 1: Japanese Laid-Open Patent Publication No. 2007-143497

The fertilized egg system of the goldfish into which the gene expression vector is introduced may be a dry-guided method in which a fertilized egg obtained by a natural person, a pre-mixed unfertilized egg from a female goldfish, and a sperm extracted from a male goldfish are added and a feeding water is added thereto to promote insemination ( The person modulated by artificial insemination by reference to methods 2) and the like.

Unfertilized eggs and sperm can be directly used for artificial insemination after extraction, and can also be used by keeping the breeding conditions in the parental fish separately and experiencing the time after storage. For example, artificial insemination may be carried out using a parental breeding fish under the conditions of breeding (about 15 to 26 ° C) or a viable water temperature (0 to 40 ° C) for 6 hours or more.

The zebrafish used in the past is also the same as the goldfish. The unfertilized egg and the sperm can be obtained by extracting the abdomen from the mature male and female fish, and then using the dry guiding method (Reference 2) for artificial insemination. However, the insemination time of unfertilized eggs and sperm extracted from zebrafish is only a few minutes short, so it cannot be stored separately as goldfish. Moreover, due to the small size of the fish, the damage to the fish body is extremely great at the time of extraction, and even in a highly skilled manner, the fish species are often killed. Therefore, it is impossible to repeatedly take eggs and collect sperm, so the productivity of fertilized eggs is extremely inferior compared with natural spawning.

Moreover, since the fertilized egg of the zebrafish is a separate type of submerged egg, it is different from the fertilized egg of the goldfish that adheres to the fallen egg, and has no biological characteristics attached to plants, rocks, plastics, etc. It is difficult to introduce problems with the target gene.

Reference 2: Green Book "Aquaculture Lecture" 4 Aquaculture, Takashima Shifu, Yu Shenggong Editor (ISBN4-89531-434-0) "Insemination Method", pp. 182-183

In the "gene-transferred goldfish" of the present invention, the goldfish into which the target gene is introduced is genetically a fish which is derived from the carp to the genus Aphisidae, such as a fish having a body color and/or body shape which has different appearance characteristics from squid. Yes. For example, it has red, red, white, tri-color, black, brown, cyan, cherry, aurora, albino, transparent scale, and dandelion; it has a squid type, a gilt type, a lion head type, and a blue shou type. Other types of appearance include sarcoma, convex eyes, ocular eyes, hydrangea, pearl scales, butterfly tails, peacock tails, etc., and goldfish such as those characterized by their own performance or multiple features.

Such goldfish can be enumerated, for example, gold, gold, Zhu Wenjin, Huixing goldfish, Tosajin, Dijin, Chaotianyan, Hydrangea, Pearl Scale, Osaka Lanshou, Butterflytail, Panda Goldfish, Sakurajin, Dragon Eye, Five Hua Jin Jin, Lan Shou, Dutch Lion Head, Dong Jin, Jiang Yu Jin, Bin Jin, Dan Ding, Qing Wen Yu, Zi Gao Tau and Nanjing, etc., and also include the new species of these mating, mutation and selection breeding. Goldfish and so on.

The method for producing a gene-transformed fish of the present invention may be any one of the steps of including a step of introducing a gene expression vector into which a target gene is introduced into a fertilized egg of a goldfish. Furthermore, the procedure of confirming the expression of the introduced target gene is also included.

In the step of "introducing a gene expression vector into a target gene into a fertilized egg of a goldfish", the gene expression vector can be introduced into a fertilized egg using a conventional method such as microinjection.

Hereinafter, the present invention will be described in more detail by way of examples, but the invention is not limited thereto.

[Examples] 1. Modulation of goldfish fertilized eggs

Fertilized eggs are fertilized eggs prepared from male and female adult fish of goldfish.

The unfertilized eggs and spermatozoa extracted from the male and female gold of 2 years old were subjected to artificial insemination by dry-guided method, and then the adhesion of the fertilized egg of the goldfish adhered to the egg was adhered and coexisted with the breeding water to a diameter of 90 mm. In a plastic dish. Further, the same artificial insemination is performed on the unfertilized egg and the sperm extracted from the male and female transparent goldfish of one year old, and the obtained fertilized egg is stored.

2. Production of gene expression vector

Three types of gene expression vectors were produced.

A. pXI-EGFP-pZex-DsRed vector

The vector is a gene expression vector of pXI and pZex which are incorporated as a promoter. The pXI-EGFP vector incorporated into EGFP (SEQ ID NO: 2) downstream of pXI (SEQ ID NO: 1) and the DsRed (SEQ ID NO: 1) downstream of pZex (SEQ ID NO: 4) The pZex-DsRed vector of 5) is ligated into a string (series) to produce a gene expression vector.

The pXI-EGFP-pZex-DsRed vector of the present invention was produced as follows.

1) Manufacture pBSIISK(-)SK-Bgl/KS-Nde (Fig. 1).

The pBSIISK(-)SK-Bgl/KS-Nde is a carrier for subcloning constructed for tandem vector production. BglII was inserted between NotI and XbaI at the multiple selection site (MCS: multicloning site) of pBluescriptIISK(-) (Stratagene), and was further produced by inserting NdeI between XhoI and KpnI.

2) Manufacture of pXI-EggE07F09 (SfiI) (Fig. 2).

The pXI-EggE07F09 (SfiI) is a vector in which the S-Iin intron of the research vector-pXI-GFP (Reference 3) has two SfiI vectors inserted downstream, and it is expected that the target gene can be set at two SfiI positions. Recombination is easier. The manufacture of pXI-EggE07F09 (SfiI) was carried out as follows.

a. Manufacture pBS11(SK-)-EggE07F09.

The pTripleEx2-EggE07F09 of the cDNA of the zebrafish ribosomal protein L35 (NCBI accession no. NP775340) was selected from the multiple selection sites of the selection vector pTripleEx2 (TAKARA BIO) to treat the restriction enzymes EcoRI and SalI. The EggE07F09 cDNA region was cut. pBII(SK-)-EggE07F09 was produced by recombining pBluescriptIISK(-) treated with restriction enzymes EcoRI and SalI via the EcoRI/SalI position.

b. Production of pZex-EGFP-His (B).

The zebrafish zHel promoter is about 1000 bp (hereinafter referred to as zHe1-1000 bp; Sequence Listing SEQ ID NO: 3), which is recombined with the promoter of XhoI/BamHI and pX1-EGFP (Reference 3), and then treated with XhoI/EcoRI via Klenow Fragment. (TAKARA BIO) The ends were smoothed, and the promoter region was deleted to about 300 bp by self-ligation to prepare pZex (SEQ ID NO: 4). Further, a His tag was added to the C-terminus of the amino acid sequence of EGFP to produce pZex-EGFP-His (B).

c. Manufacture pZex-EggE07F09 (SfiI) (Fig. 3).

The region containing EggE07F09 was cut with BamHI/SalI from pBSII(SK-)-EggE07F09. This was recombined with pZex-EGFP-His (B) via BamHI/SalI to produce pZex-EggE07F09 (SfiI).

d. Manufacture pX1-EggE07F09 (SfiI).

The polyA addition signal region (Signal region) derived from pZex-EggE07F09 (SfiI) of EggE07F09 and +SV40 was cleaved with BamHI/SalI. This was recombined with pX1-EGFP via BamHI/NdeI to produce pX1-EggE07F09 (SfiI).

3) Manufacture of pXI-1000pro-DsRed (ΔBamHI) (Fig. 4).

pXI-1000pro-DsRed (△BamH1) recombines the zebrafish zHe1 promoter by about 1000 bp via the promoter of XhoI/BamHI and pXI-EGFP, and then transfects EGFP via pDsRed-Monomer (Clontech) with pDsRed-Monomer and BamHI/NotI. The vector for recombination. After treatment with BamHI, the ends were smoothed by Klenow Fragment (TAKARA BIO), and the recognition sequence of BamHI of the vector was deleted by auto-ligation.

4) Make pZex-DsRed (ΔBamH1)/pBSIISK(-)SK-Bg1/KS-Nde (Fig. 5).

pZex-DsRed(?BamH1)/pBSIISK(-)SK-Bg1/KS-Nde is the pBSIISK(-)SK-Bg1 manufactured by the above 1) by pX1-1000pro-DsRed (?BamH1) manufactured by the above 3) /KS-Nde is a vector for recombination via EcoRI/NdeI.

5) Manufacture of pXI-EggE07F09 (SfiI)-pZex-DsRed (Fig. 6).

pXI-EggE07F09(SfiI)-pZex-DsRed is a Bg1II/NdeI fragment of pXI-EggE07F09 (SfiI) manufactured by the above 2) and pZex-DsRed (ΔBamH1)/pBS11SK(-)SK- manufactured by the above 4). The BamHI/NdeI fragment of Bg1/KS-Nde was produced by ligation.

6) Production of pXI-EGFP (SfiI)-pZex-DsRed (Fig. 7).

The plastid of pXI-EggE07F04(SfiI)-pZex-DsRed's EggE07F09 produced by the above 5) was recombined via SfiI and EGFP.

B.pZex-EGFP vector

A vector into which EGFP (SEQ ID NO: 2) was incorporated downstream of pZex (SEQ ID NO: 4) was produced.

That is, zHe1-1000bp (SEQ ID NO: 3) was recombined via the promoter of XhoI/BamHI and pX1-EGFP (Reference 3), and then treated with XhoI/EcoRI to smooth the ends via Klenow Fragment (TAKARA BIO). The promoter region was deleted to about 300 bp by auto-ligation to prepare pZex-EGFP having pZex (SEQ ID NO: 4).

C. pZEF1-EGFP vector

This vector is substituted with pZEF1 (SEQ ID NO: 6) as a promoter in the promoter region of pXI-EGFP vector (pXI; sequence listing SEQ ID NO: 1) and is integrated into EGFP (SEQ ID NO: 2 in the sequence listing). Gene expression vector.

The pZEF1-EGFP vector of the present invention was produced as follows.

1) Modulation of zebrafish genome DNA

The zebrafish genomic DNA was prepared according to Reference 4. That is, about 100 zebrafish (golden type) juveniles (14dpf) after preparation for hatching (after 48hpf) and Proteinase K (Wako 160-14001) 0.2μg/mL (dissolved in SET buffer (10mM-Tris pH7. 5,5mM-EDTA, 1% SDS)). Among them, 50 juveniles were suspended in 2 mL of 0.2 μg Proteinase K solution, and incubated at 37 ° C for 2 hours, then shaken overnight to keep the shaker shaken and incubated at room temperature.

To the above-prepared zebrafish enzyme decomposition sample (500 μL), the same amount of PCI (phenol: chloroform: mixture of isoamyl alcohol = 25:24:1) was added, followed by mixing with a relaxation oscillator. After centrifugation at 20,000 x g for 20 minutes at room temperature, 300 μL of the supernatant was recovered using a wide-mouth micropipette. 30 μL of 3 M sodium acetate and 825 μL of 100% ethanol were added thereto, mixed, and allowed to stand at -80 ° C for 30 minutes, and then centrifuged at 20,000 × g for 40 minutes at 4 ° C. The obtained granules were washed twice with cold 70% ethanol, and then dissolved in 100 μL of 0.1×× to prepare zebrafish genomic DNA.

Reference 4: Development 103. 403-412 (1988)

2) Modulation of the promoter sequence

The zebrafish genomic DNA prepared by the above 1) is isolated from the base sequence of pZEF1 by PCR. PCR was carried out under the conditions described in Table 1 using PrimeSTAR of TAKARA using the primers of Sequence Listing No. 7 and SEQ ID NO: 8.

The base sequence of the amplified DNA fragment was determined using a DNA sequencer (manufactured by Beckman). Thus, the base sequence described in Sequence Listing No. 6 of the Sequence Listing is a promoter region (pZEF1) of about 1 kb upstream of the EF1a gene.

Both ends of the DNA fragment amplified by PCR were smoothed, and the primer described in Sequence Listing No. 9 further had a position of Sse83871 (manufactured by TAKARA BIO). The 5' end of the DNA fragment amplified by this PCR becomes a smooth end, and the 3' end can be modulated to the Sse83871 recognition position. Therefore, it is possible to incorporate a vector in which both ends are modulated into a smooth end of PmeI and a position of Sse83871.

3) Manufacture of carriers

The research vector pzHel-1000pro-EGFP (SfiICB) (i) (Fig. 8) was constructed by using pXI-EGFP as a substrate.

The base sequence between the NotI position at position 111 of the base sequence of pzHel-1000pro-EGFP (SfiICB) (i) to the AsiSI position at position 2362 of the base sequence is identical to that of pXI-EGFP. From the AsiSI position at position 2362 of the base sequence of pzHel-1000pro-EGFP(SfiICB)(i) to the position between the NotI positions at position 111 of the base sequence, zHel-1000bp (SEQ ID NO: 3), EGFP (SEQ ID NO: 3) 2) The SV40 polyA signal (SEQ ID NO: 9) is arranged in the order of 2146 bp (SEQ ID NO: 10). In addition, PmeI position and Sse83871 position, SfiI position and SfiI position, ClaI position and XbaI position existed upstream and downstream of zHel-1000bp, EGFP and SV40 polyA signal, respectively.

Then, the promoter sequence (promoter of zebrafish zHel; SEQ ID NO: 3) linked to the position of Pmel-Sse83871 of the vector was removed, and the promoter sequence of the above 1) was added to prepare a ZEF1-EGFP vector (p. 9 map).

It was confirmed that the three vectors of A to C produced above were expressed in the initial embryo of zebrafish.

3. Gene introduction

Gene introduction was carried out by microinjection to introduce these gene expression vectors. That is, the gene expression vectors of the above A and B were prepared to be 100 ng/uL, filled into a capillary tube for microinjection made of glass, and microinjected into the fertilized egg immediately after fertilization.

4. Confirmation of gene expression

The fertilized eggs after microinjection were cultured at 20 ° C, and the expression of the fluorescent protein was observed using a fluorescence microscope as appropriate.

As a result, as shown in Fig. 10, the gene expression vector (pXI-EGFP-pZex-DsRed vector) introduced into A. was confirmed to have EGFP expression throughout the whole embryo introduced into the embryo after 72 hours of fertilization, and The hatching gland specifically has DsRed performance. Further, in the fertilized egg derived from the transparent goldfish, the gene expression vector (pXI-EGFP-pZex-DsRed vector) introduced into A. is shown in Fig. 11, and it was confirmed that the embryo was introduced into the early embryo after 72 hours of fertilization. There are EGFP manifestations throughout the body, and the hatching glands specifically have DsRed performance.

Further, as shown in Fig. 12, the gene expression vector (pZex-EGFP vector) introduced into B. was confirmed to have EGFP expression throughout the embryo introduced into the initial embryo 24 hours after fertilization, and 72 hours after fertilization. The hatching gland has a specific expression. Moreover, after 96 hours of fertilization, the EGFP of the hatching gland showed attenuating.

Furthermore, as shown in Fig. 13, the gene expression vector (pZEF1-EGFP vector) introduced into C. was confirmed to have EGFP expression throughout the whole embryo introduced into the initial embryo 48 hours after fertilization.

From the above results, it can be seen that the genetically-transferred goldfish can be produced according to the production method of the present invention.

[Industrial availability]

According to the method for producing a genetically-transferred goldfish according to the present invention, a gene-transferred goldfish into which various genes are introduced can be produced. By introducing a specific gene, it is possible to investigate the effects of the gene on goldfish and to make useful proteins. The protein produced by the gene-transferred goldfish of the present invention can be easily extracted from serum, and thus the protein can be easily and largely obtained as compared with the conventional gene-transformed fish.

<110> TAMARU, Yutaka AKIYAMA, shinichi

<120> Transgenic goldfish.

<130> TAM002

<150> JP 2009-107344

<151> 2009-4-27

<160> 10

<170> Patentln version 3.1

<210> 1

<211> 1161

<212> DNA

<213> Xenopus laevis

<400> 1

<210> 2

<211> 720

<212> DNA

<213> Victorian Jellyfish (Aequorea victoria)

<400> 2

<210> 3

<211> 1000

<212> DNA

<213> Zebrafish (Danio rerio)

<400> 3

<210> 4

<211> 290

<212> DNA

<213> Zebrafish (Danio rerio)

<400> 4

<210> 5

<211> 678

<212> DNA

<213> Sea Anemone

<400> 5

<210> 6

<211> 918

<212> DNA

<213> Zebrafish

<400> 6

<210> 7

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer (zEf1-alpha_1054bpU)

<400> 7

<210> 8

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Primer (zEf1-alpha_rest1D)

<400> 8

<210> 9

<211> 257

<212> DNA

<213> SV40 virus

<400> 9

<210> 10

<211> 2146

<212> DNA

<213> Zebrafish

<400> 10

Fig. 1 is a diagram showing pBluescriptIISK(-)SK-BgI/KS-Nde (Example).

Figure 2 is a diagram showing the pXI-EggE07F09 (SfiI) (Example).

Figure 3 is a diagram showing the pZex-EggE07F09 (SfiI) (Example).

Figure 4 is a diagram showing pXI-1000pro-DsRed (?BamHI) (Example).

Figure 5 is a diagram showing pZex-DsRed(?BamHI)/pBSIISK(-)SK-Bgl/KS-Nde (Example).

Figure 6 is a diagram showing pXI-EggE07F09(SfiI)-pZex-DsRed (Example).

Figure 7 is a diagram showing pXI-EGFP(SfiI)-pZex-DsRed (Example).

Figure 8 is a diagram showing the pzHel-1000pro-EGFP (SfilCB) (i) (Example).

Figure 9 is a diagram showing the ZEF1-EGFP vector (Example).

Fig. 10 is a diagram showing the expression of the introduced gene (Example).

Fig. 11 is a diagram showing the expression of the introduced gene (Example).

Fig. 12 is a view showing the expression of the introduced gene (Example).

Fig. 13 is a diagram confirming the expression of the introduced gene (Example).

No component symbol description

Claims (10)

A method for producing a gene-transferred goldfish, comprising the step of introducing a gene expression vector incorporated into a target gene into a fertilized egg of a goldfish, wherein the gene expression vector comprises pXI as a promoter (SEQ ID NO: 1) and pZex (SEQ ID NO: 4), or pZEF1 (SEQ ID NO: 6) as a promoter. The method for producing a gene-transferred goldfish according to the first aspect of the invention, wherein the gene expression vector comprises pXI (SEQ ID NO: 1), pZex (SEQ ID NO: 4), and pZEF1 (SEQ ID NO: 4) as a promoter 6) Any two or more. The method for producing a gene-transferred goldfish according to the second aspect of the patent application, wherein the two or more of pXI (SEQ ID NO: 1), pZex (SEQ ID NO: 4), and pZEF1 (SEQ ID NO: 6) are Arrange in series. The method for producing a genetically-transferred goldfish according to claim 1 or 2, wherein the target gene system is expressed throughout the body. For example, the method for manufacturing a genetically-transferred goldfish according to claim 1 or 2, wherein the goldfish is selected from the group consisting of gold, gold, Zhu Wenjin, Huixing goldfish, Tosajin, Dijin, Chaotianyan, Hydrangea, and pearl scale. Osaka Lanshou, Butterfly Tail, Panda Goldfish, Sakura Jin, Dragon Eye, Wuhua Jinjin, Lanshou, Dutch Lion Head, Dongjin, Edo Jin, Binjin, Danding, Qingwenyu, Zi Gaotou and Nanjing Zhongzhi One. A gene expression vector, which is a gene expression vector used in a method for producing a gene-transferred goldfish, which comprises pXI as a promoter (sequence list) SEQ ID NO: 1) and pZex (SEQ ID NO: 4), or pZEF1 (SEQ ID NO: 6) as a promoter. A gene expression vector according to claim 6 which contains, as a promoter, pXI (SEQ ID NO: 1), pZex (SEQ ID NO: 4), and pZEF1 (SEQ ID NO: 6) . The gene expression vector of claim 7, wherein two or more of pXI (SEQ ID NO: 1), pZex (SEQ ID NO: 4), and pZEF1 (SEQ ID NO: 6) are arranged in series. The gene expression vector of claim 6 or 7, wherein the gene expression vector is introduced into a fertilized egg of a goldfish. For example, the gene expression vector of claim 6 or 7, wherein the goldfish is selected from the group consisting of gold, gold, Zhu Wenjin, Huixing goldfish, Tosajin, Dijin, Chaotianyan, Hydrangea, Pearl Scale, Osaka Lanshou, Butterfly tail, Panda goldfish, Sakura Jin, Longjing, Wuhua Jinjin, Lanshou, Dutch Lion Head, Dongjin, Edojin, Binjin, Danding, Qingwenyu, Zi Gaotou and Nanjing.