JP2002125517A - Method for creating embryo cell clone fishes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for creating a clone of fish, being a diploid, having fertility. SOLUTION: This method for creating a clone of fish is characterized in that the method has a process for irradiating a nucleus recipient cell with X- rays, a process for transplanting the nucleus of a nucleus donor cell into the nucleus recipient cell and a process for producing the obtained nucleus transplanted cell and the nucleus donor cell is an embryo cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing fish clones.

[0002]

BACKGROUND OF THE INVENTION Animal clones were first studied in the 1950s using frogs. Since then, research has been conducted on fish and mammals. It has become a key technology in biological and medical industry and academic research, as seen in recent sheep success [KHSCampbell et al., Natur
e, 380, 64 (1996), I. Wimut et al., Nature, 385, 810 (1997)].
At the heart of such cloning techniques is the transfer of cell nuclei from the same individual to eggs. This allows, for example,
Many clones (ie, copies) of excellent individuals can be made.

[0003] Fish is an important industrial resource. However, a method for producing the clone has not been established.
For example, attempts in goldfish and European locusts [TCTun
g et al., Scientica (Peking), 14, 1244 (1963)], an attempt in loach [KGGasaryan et al., Nature, 280, 587 (1979)],
Trial in Medaka [K. Niwa et al., Develop.Growth Diffe
r.41, 163 (1999)], an attempt in carp fish [SYYan, In
Cytoplesmic Organization Systems: A primer in deve
lopmental biology (ED.GMMalacinski), 61 (1989), S.
Y.Yan,; Nucleocytoplasmic Hybrids.Educational and C
ultureal Press, Hong Kong (1998)], etc., but the resulting clones are triploid, non-fertile or sexually mature adults. There are problems such as not being able to obtain [K. Niwa et al.,
Develop. Growth Differ, 41, 163 (1999), K. Niwa et al., Clon.
ing2, 23 (2000)].

[0004]

SUMMARY OF THE INVENTION In view of these circumstances, an object of the present invention is to provide a method for producing a diploid and fertile fish clone.

[0005]

Means for Solving the Problems As a result of earnest research for achieving the above object, the present inventors have found the following means. That is, a method for producing a clone of a fish, comprising the steps of irradiating a nuclear acceptor cell with X-rays, transplanting a nucleus of a nucleus-providing cell into the nuclear acceptor cell, and generating the obtained nuclear transfer cell. And wherein the nucleus-providing cell is an embryonic cell.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for producing clones in fish. Specifically, the method is characterized in that nucleus-receiving cells are irradiated with X-rays to enucleate, and nuclei of nucleus-providing cells collected from a donor are transplanted to the nucleus-receiving cells.

The method of the present invention will be described with reference to FIG. The embryo cells 3 are dissociated from the embryo 2 obtained from the donor 1. On the other hand, oocytes 6 are collected from the recipe end 5. Oocyte 6
Then, the embryo cells 3 are implanted into the oocytes 6 using the micropipette 4. Thereafter, the nuclear transfer body 8 is obtained while maintaining the inside of the incubator.

The fish that can be used in the present invention may be, for example, research fish such as medaka, tilapia and vera, and edible fish such as salmon, trout, yellowtail and tuna, but are not limited thereto. , Any fish species. The method of the present invention can be used for fish in general.

As used herein, the term "nucleus donor cell" refers to a cell for providing a nucleus, and the individual from which the cell originates is referred to as a "donor". The nucleus providing cell used in the method of the present invention may be a cell having totipotency, and for example, an embryonic cell is preferable. The donor to be used may be any fish species, and may be a fish into which a desired gene has been introduced in advance.

[0010] As used herein, the term "nuclear recipient cell" refers to a cell that is provided with a nucleus, and the individual from which the cell originates is referred to as a "recipient." The nuclear recipient cell may be any egg or oocyte of the above fish. Also, the donor and the recipient need not necessarily be of the same species.

[0011] X-ray irradiation of the nuclear recipient cells is performed to enucleate the nuclear recipient cells. For example, about 100-
About 200 Gy X-rays may be applied at a dose rate of about 8.5 Gy per minute for about 11.8 to about 23.5 minutes. Further, each condition may be appropriately changed according to the X-ray facility to be used.

When embryo cells are used as nucleus-providing cells, for example, blastula stage embryos may be used. The transplantation, for example, after dissociating the embryo into individual cells by a general method known per se such as pipetting, sucked into a thin glass tube,
What is necessary is just to transplant to the animal pole side of the cytoplasm of a nuclear recipient cell.

[0013] The cells after nuclear transfer are prepared in a balanced salt solution for about 1 hour.
Maintain at 6 ° C. to about 18 ° C. for 1 day, then at 26 ° C. until hatch. Here, conditions such as the composition of the balanced salt solution are as follows:
What is necessary is just to select according to the fish egg used.

[0014]

EXAMPLES Examples of nuclear transfer using medaka (Oryzias latipes) embryo cells are described below. In experiment 1, EF-
Inbred HNI-I transfected with 1α-A / GFP
Was used as a donor, and outcrossed Himedaka (hereinafter referred to as O
Nuclear transfer was performed using R as a recipient. In Experiment 2, nuclear transfer was performed using OR into which β-actin / GFP-N had been transfected as a donor and OR without transfecting had been used as a recipient. Furthermore, mating tests were performed on the nuclear transplanted fish that had reached the adult fish stage to evaluate fertility.

[Experimental Materials and Experimental Methods] Donor (1) Inbred strain H having EF-1α-A / GFP gene
NI-I Inbred HNI-I (hereinafter, also simply referred to as HNI-I)
Indicates the wild-type body color has a PGM ^a is phosphoglucomutase allo Zyme marker [Y.Wakamatsu
Et al., Mol. Marine Biol. Biotech. 2 (2), 325 (1993);
Taguchi, Y., FishBiol. J. MEDAKA 8, 11 (1996)]. Wild type color is a dominant trait to scarlet color. Also, HN
II matures sexually within 2.5 months.

In Experiment 1, the transfected HNI-I used as a donor was a medaka elongation factor 1α-A
It has a green fluorescent protein gene (hereinafter referred to as EF-1α-A / GFP) driven by a gene promoter [Kinos
hita, M., Kani, S., Ozato, K. and Wakamatsu, Y., Develop.
Growth Differ. 42,469 (2000)]. Specifically, the gene was introduced by the following method.

First, medaka translation elongation factor 1α-A (EF-1
α-A) gene [M.Kinoshita et al., Fisheries Sci. 65, 765 (199
9)] (ca2.8kbp) was amplified by PCR. To prepare pEF-1α-A / GFP, pCMX / GFP-1 [H. Ogawa and K. Umezono, A.
CMV in cta Histochem. Cytochem. 31, 303 (1998)]
Was replaced with the promoter region. Prior to the first cleavage, the plasmid pEF-1α-A / GFP was microinjected into the cytoplasm of fertilized eggs of HNI-I medaka. E
Homozygotes for F-1α-A / GFP were generated by a crossing test of transgenic fish.

FIG. 3A shows a photograph of a transgenic fish having such EF-1α-A / GFP. FIG. 3A was taken under visible light. GFP fluorescence in this transgenic fish was observed throughout the body from the early sac embryo to the 5-day embryo stage. Furthermore, throughout subsequent developmental stages, GFP fluorescence was observed in various tissues except muscle (FIG. 3B). In this nuclear transfer, embryo cells were used as nucleus donor cells.

(2) OR experiment having β-actin / GFP-N gene In the experiment 2, the transfected OR used as a donor was the medaka β-actin gene promoter having a nuclear localization signal as described above. Are homozygous for the GFP gene (hereinafter referred to as β-Act / GFP-N). Gene transfer is performed as follows.

First, using pOBA [S. Takagi et al., Mo
l. Mar. Biol. Biotech., 3, 192 (1994)], and the medaka β-actin gene promoter region (ca. 3.7 kbp) was amplified by PCR. Next, in order to prepare pβ-Act / GFP-N, the promoter region of pCMX / GFP-1 was
By substituting the promoter region of the medaka β-actin gene, the SV40-derived nuclear localization signal
Linked to the 3'-end of the gene [Yamauchi, M. et al., J. Exp.
ol. Printing]. Before the first cleavage, plasmid pβ-Act
-GFP-N was microinjected into the cytoplasm of the OR fertilized egg. Homozygotes for β-Act-GFP-N were created by a transgenic fish mating test.

FIG. 3F shows a transgenic fish into which β-Act / GFP-N has been introduced. Here, FIG. 3F is taken under visible light. GFP fluorescence at the OR with β-Act / GFP-N was first observed in the somites of 2-day embryos. However, strong fluorescence was restricted to the skeletal muscle (FIG. 3G). In this nuclear transfer, embryo cells were used as nucleus donor cells.

2. Recipient In Experiment 1 and Experiment 2, both ORs without gene transfer were used as recipients. FIG. 2 shows a photograph of the OR. In this nuclear transfer, an oocyte was used as a nuclear receptor cell.

[0023] OR is an outbred system medaka of the body color is scarlet, which has the PGM ^b is phosphoglucomutase allo Zyme marker [Y.Wakamatsu et al, Mol.Ma
rineBiol. Biotech. 2 (2), 325 (1983)]. It also sexually matures in 1.5 months.

3. Rearing conditions The rearing of the above fish was performed under the condition of 26 ° C. in which lighting was controlled by a light-dark cycle consisting of a light period of 14 hours and a dark period of 10 hours. The feed is tetramine (TetraMin, TetraWerke, Me
lle, Germany) and fed a brine shrimp larva (Ocean Star International, Snowville, NH, USA).

4. Preparation of nucleus donor cells The stage of embryo development was determined according to the stage of Iwamatsu development [Iwamat
su, T., Zool. Sci. 11, 825 (1994)].

15-20 early blastula stages (ie, stage 10)
Blastoderm was recovered from donor embryos of Wakamatsu by the method of Wakamatsu et al. [Wakamatsu, Y., Sasado, T. and Ozato, K., Mol. Mar. Bi.
ol. Biotechnol. 3, 185 (1994), K. Niwa et al., Develop.
h Differ, 41, 163 (1999), K. Niwa et al., Cloning 2, 23 (200
0)]. In a 1.5 mL test tube, Ca ²⁺ , Mg
^The collected blastoderm was dissociated into individual cells by pipetting in 2 ^{+ -free} phosphate buffered saline (CMF-PBS). The resulting cell suspension is treated with 50 × g for 5 minutes 4
Centrifuged at ° C.

The cell pellet was prepared by changing the composition of a transplantation buffer for Xenopus laevis (hereinafter referred to as TB).
[Gurdon, JB, J. Emb.
ryol.Exp.Morphol.36,523 (1976)]. The composition of TB is as follows: sucrose of 0.25 mol / L, 120
mmol / L NaCl, 0.5 mmol / L spermine trihydrochloride, 0.15 mmol / L spermine tetrahydrochloride, and 15 mmol
1 / L HEPES (pH 7.3). To make it easier to identify the cells during the microinjection step, a final concentration of 0.005-0.01% neutral red (Molec
(ular Probes, Eugene, OR, USA).
Specifically, the staining was performed by adding a stock solution of neutral red (concentration: 1%) to the cell suspension. The cells were maintained at 4 ° C. until use.

5. Preparation of Nuclear Receptor Cells Female fish that had been laying daily were transferred to an isolation tank and isolated on the day before the experiment. On the day of the experiment, they were sacrificed within 3-4 hours from the beginning of the light period. Collect the matured oocytes ovulated into the ovarian cavity and use a 35 mm plastic dish for suspension culture
(Sumilon, Sumitomo Bakelite) at 18 ° C. until use. This culture dish contains a balanced salt solution for medaka (hereinafter referred to as BSS) to which 100 U / mL penicillin and 100 μg / mL streptomycin have been added [Iwa
matsh, T., J. Exp. Zool. 228, 83 (1983)].

6. Enucleation by X-ray irradiation For mature oocytes collected from OR, 100-20
X-ray irradiation was performed at 0 Gy to enucleate. Specifically, it is as follows. The oocytes were placed in BSS in a 60 mm plastic dish coated with 0.1% bovine serum albumin (BSA). The BSS was poured to a position 0.5 mm higher than the upper edge of the oocyte.

The X-ray irradiation was performed using an X-ray apparatus (model OM-100RE, OHMI
C, Tokyo) at 80 kV and 8 mA. Oocytes were irradiated at a dose rate of 8.51 Gy per minute and a total X dose of 100 Gy or 200 Gy.

7. Nuclear transfer was performed using a method basically similar to the conventional method [K. Niwa et al., Develop.Growth Differ, 41, 163 (1999), K.
Niwa et al., Cloning 2, 23 (2000)]. The procedure of the transplantation is as shown in FIG. 1 (FIG. 1). Implantation was performed under a stereomicroscope (MZAPO, Leica, Heerbrugg, Switzerland) with a magnification of 80x. 60 mm plastic dish filled with BSS containing antibiotics (Falcon, Becton-Beckington, Lincoln Park, NJ
The nuclear recipient cells (ie, nuclear recipient eggs) were fixed in 1 mm wide V-shaped grooves formed on 2% agar in J. USA. Nuclear donor cells were also dispersed on the same agar plate.

On a cooling plate placed on the stage of a stereo microscope
(Thermo Plate; Tokai Hit) and the temperature of the agar plate was maintained at about 10 ° C. The micropipette for transplantation is 1m in outer diameter using a puller (PN-3, Narishige).
It was prepared by drawing a glass capillary of m.
The inner diameter of the opening of the micropipette is about 20-30 μm
Which is equal to or slightly smaller than the diameter of the nucleus donor cell.

A hydraulic injector (CellTram Oil, NO-202, Narishige) connected to a three-dimensional micromanipulator (NO-202, Narishige)
(Eppendorf, Hamburg, Germany), by partially modifying the method of transferring embryo cells to embryos using the micropipette [Wakamatsu, T., Hashiomoto, H., Kinoshi
2, 325 (1993)], and one nucleus donor cell was aspirated and transplanted from the ovum of a nuclear recipient egg into the cytoplasm of the animal pole.

Each of the treated eggs was transferred to a 24-well plastic plate (Sumilon, Sumitomo Bakelite) filled with BSS containing 2 ppm of methylene blue.
The plates were incubated for the first 24 hours at 18 ° C, then at 26 ° C and observations were made daily until hatch. The hatched fry was prepared by the method of Wakamatsu et al. (Wakamatsu, T. (199
3) See above).

The identification numbers given to the two fry that hatched in Experiment 1 were 1NT1 and 1NT2, respectively.
In Experiment 2, the identification numbers assigned to the five fry that hatched and grew were 2NT1, 2NT2, and 2NT, respectively.
3, 2NT4 and 2NT5 (that is, 2NT1-2NT5).

Nuclear transplants that had matured to the adult stage were used for mating studies and then sacrificed for use of the tissue for further testing.

8. Detection of GFP Fluorescence GFP was detected by a stereo microscope (FLI
I, Leica) was used to observe the fluorescence of GFP in embryos and fish. The fluorescence image was taken using a microscope camera (MPS60, Leica) attached to the stereo microscope.

9. Analysis phosphoglucomutase by electrophoresis of allozymes (hereinafter referred to as PGM) allozyme analysis was performed on F ₁ generation of 1NT1 the five cases. PGM
Allozyme analysis by electrophoresis was performed using a partially modified conventional method (Wakamatsu et al. (1993) supra).

That is, the liver tissue was treated with 30 μmol / L.
Protein inhibitor mixture containing (p-amidophenyl) methanesulfonyl fluoride (Wako Pure Chemical Industries), 100 μg / mL trans-epoxysuccinyl-L-leucylamide (4-guanidino) butane (Sigma) and 1 mmol / L EDTA And homogenized with an equal volume of distilled water.

The homogenized tissue was treated for 4 minutes for 4 minutes.
Centrifugation was performed at 18,000 xg at a temperature. BPB-glycerin solution (0.02% bromophenol blue and 3% in distilled water)
(Containing 0% glycerin) was added to the supernatant.
This sample (4 μL) was placed on a 10% polyacrylamide gel and electrophoresed at a constant current of 20 mA for 1 hour at 4 ° C.

After electrophoresis, the gel was treated with 6 mg / mL
Glucose monophosphate, 2 mg / mL MgCl ₂ ,
0.1 mg / mL NADP, 0.1 mg / mL 3-
(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-tetrazolium bromide, 0.025 mg / mL phenazine methosulfate and 1 U / mL glucose-6-
0.1 mol containing phosphate dehydrogenase
/ L Tris-HCl solution (pH 7.1) was used to stain PGM for 5 minutes at 37 ° C.

10. Detection of GFP gene The GFP gene was detected as follows. First, the method of Brin and Stafford [N. Blin and DMSta
fford, Nucleic Acid Res. 3, 2303 (1976)], genomic DNA was extracted from adult fins or hatched fry. PCR was performed with 0.5 unit of ExTaqDN.
A (Takara), 100 nM of each primer, and about 50 ng of DNA as a template were used in 20 μL.

A primer specific to the GFP gene [forward chain: 5'-TGCCACCTACGGCAAGCTGA-3 '(this is represented by SEQ ID NO: 1)
) And the reverse chain: 5′-TGTTGCCGTCCTCCTTGAAG-3 ′ (this is SEQ ID NO: 2)] and EF-1α-A / G
An FP transgene (expected size: 299 bp) was detected. Primer W4 [ie, a sequence specific to the β-actin gene promoter region (5′-ACAAGAGAATGCAGCCCA-3 ′, which is referred to as SEQ ID NO: 3)] and SAH-Rev [ie, a sequence specific to the GFP gene (5 '-TGAAGTTGTACTCCAGCTTG-3', which is referred to as SEQ ID NO: 4)] to obtain β-Act / GFP
The -N transgene (expected size: 652 bp) was detected.

To confirm the success of DNA extraction and subsequent PCR, the endogenous EF-1α-A gene was replaced with Medaka EF
-1α-A gene specific primer [5'-CAGGACGTC
TACAAAATCGG-3 '(this is SEQ ID NO: 5), 5'-AGCTC
GTTGAACTTGCAGGCG-3 '(this is SEQ ID NO: 6)]. The expected size of the PCR product is 519 bp
Met.

The PCR parameters are as follows: after 3 minutes at 94 ° C., followed by 30 seconds at 94 ° C.
A cycle of 55 ° C. for 45 seconds and 72 ° C. for 1 minute
32 cycles for EF-1α-A / GFP and 35 cycles for β-actin / GFP-N.

All PCR products were electrophoresed on a 1.5% agarose gel. Each nuclear transplant and 13 from 15 cases of _{F 1} generation derived therefrom, as well as 1NT1 and 2N
It was tested for _{F 2} generation fish 10 cases derived from T1.

11. All the nuclear transfer member reaches the study adult stage of chromosome number, F ₁ generation individuals 5 from each nuclear transfer member in the first and second both experiments
In each case, the number of chromosomes was examined at the adult stage. 1NT
In No. 1, tissue fragments of the caudal fin were finely minced in CMF-PBS. Then, 10% fetal bovine serum (Gibco), 2% carp serum [Use carp cultivated within Tokyo Fisheries University] in one well of a plastic 4-well dish (Nunc) coated with 0.1% gelatin solution Prepared by Dr. N. Okamoto of the university], 100 U / mL penicillin and 10
Leiboviz's L-15 medium (Leiboviz's L-15medium, Gib.) Containing 0 μg / mL streptomycin and diluted to 90% with Milli-Q water to match the osmotic pressure of fish.
co.) was added, and the tissue pieces were cultured therein.

The culture was grown in a larger culture dish (diameter 3).
Fibroblasts grown from the fragment were subcultured stepwise using a 5 mm (Falcon). Cells up to the fourth generation were treated with colcemide at a final concentration of 1 μg / mL for 4 hours. And 2NT5 from 2NT1, in the _{F 1} generation, 0.
After treatment with a 01% colchicine solution for 15 hours, the spleen and gills excised from each fish were finely minced in CMF-PBS. Chromosome specimens were prepared according to standard techniques [TL
adygina and Y.Wakamatsu, Fish Biol.J.MEDAKA.10,33
(1999)]. At least 10 cells in metaphase were examined for each fish.

12. Hybridization of Nuclear Transfer Fish Each of the adult nuclear transfer fish and their progeny were crossed with OR or inbred line medaka (hereinafter referred to as HO5), and their reproductive ability was examined. Every day eggs were collected from the abdomen of female fish and the resulting eggs were incubated at 26 ° C. in 35 mm plastic dishes filled with distilled water containing 0.5 ppm of methylene blue. Their embryonic development was observed daily under a stereomicroscope until the hatching stage. The offspring obtained as a result of the cross between the nuclear-transferred fish are the first generation of the cross.
₁ shows, indicated as F ₂ similarly mating second generation or less.

[Results] Preparation of nuclear transfer fish (1) Experiment 1: HNI having EF-1α-A / GFP
As described above, in the nuclear transfer experiment 1 using -I, OR oocytes were used as nucleus receiving cells, and EF was used as nucleus providing cells.
Nuclear transfer was performed as described above using HNI-I embryo cells having -1α-A / GFP.

In Experiment 1, 409 (69.6%) of the 588 oocytes subjected to the nuclear transfer treatment were split, and 386
(65.6%) develop at the blastula stage and an additional 29 (4.9)
%) Reached embryo body formation (one-day embryo stage). Two of them (1NT1 and 1NT2) (0.3%)
Hatched. 50 days after hatching before sexual maturation, 1NT2
Was affected by abnormal swelling of the intestine due to indigestion (Table 1).

[0052]

[Table 1]

Since the same accidental symptom occurred in the control fish at the same time, it was considered that the abnormality of 1NT2 was not due to the transplantation. 1NT2 was immature, but the presence of ovaries was confirmed, and it was confirmed that it was female. The whole body was stored at -80 C immediately after death for further studies. As with HNI-I, 1NT1 became sexually mature 2.5 months after hatching. It was female and fertile, with about 30 eggs per egg laying and of normal litter size.

(2) Experiment 2: Nuclear transfer using OR having β-Act / GFP-N In Experiment 2, as described above, an OR oocyte was used as a nucleus receiving cell, and β was used as a nucleus providing cell. -Act / GFP-N
Nuclear transfer was performed as described above using OR cells having OR.

In Experiment 2, out of the 298 oocytes treated, 209 (70.1%) cleave, 203 (68.1%) develop to the embryo stage, and 48 (68.1%) develop the embryo body stage. (One-day embryo stage). Seven (2.3%) hatched, two of which died at the larval stage. The remaining 5 cases (1.7%) (2NT1
22NT5) matured sexually in 1.5 months, as in the case of OR. They all are female, a fertility, to yield a F ₁ generation with normal litter size.

(3) G in nuclear transplanted fish and its offspring
The FP fluorescence results are shown in FIG. FIG. 3A shows EF- under visible light.
1 shows a donor transgenic fish having 1α-A / GFP.
Its body color is wild type and is 1.5 months old. FIG. 3B
Shows a fluorescence image photograph of FIG. 3A. As shown in FIG. 3B, strong fluorescence in the eyeball and weak fluorescence over the entire skin were observed. The fluorescence in the abdomen and the dot-like fluorescence in the trunk are autofluorescence of the pigment cells.

FIG. 3C is a 7 month old fluorescence photograph of 1NT1 of the nuclear transplant. Arrows are artifacts. FIG.
Is the _{F 1} generation of fish derived from the 1NT1, is an individual of the 1-month-old. Figure 3E is a F ₂ generations 1. Day embryo derived from 1NT1, (indicated by arrows in FIG. 3E) that melanophores darker pigmented seen.

FIG. 3F shows β-Act / G under visible light.
It is a photograph of a donor transgenic fish having FP-N. The individual was scarlet in color and 3 weeks old. FIG. 3G shows FIG.
It is a fluorescence image photograph of the individual F, and strong fluorescence was observed in the muscle tissue. FIG. 3H is a fluorescence photograph of a nuclear transfer obtained in the same experiment as Experiment 2. Figure 3I is a _{F 1} generation derived from 2NT1. Figure 3J is a 5 day embryos from _{F 2} generations derived from 2NT4. The yellow spots on the head and dorsal midline are the autofluorescence of the leukophores. FIG.
The bars in AD and FIGS. 3F-I represent 5 mm. The bars in FIGS. 3E and J represent 0.3 mm.

The seven nuclear transplants (1NT1, 1NT2 and 2NT)
1-2NT5) and the offspring F ₁ , F ₂ generations,
GFP was expressed in the same pattern as in the donor transgenic fish throughout the embryonic and adult stages.

2. Allozyme analysis 1NT1 and 1NT2 of PGM has a PGM ^a is allo Zyme markers of the donor type, was not a PGM ^b of the recipient type (Figure 4, Table 2).

F derived from the nuclear transplant₁In generations
Inheritance patterns of genetic markers analyzed by mating tests
did. FIG. 4 shows, in order from the left column, N
HI-I, HO5, transfected NHI-I, OR,
F between HNI-I and HO5 ₁Hybrid (HNI-I in the figure
× HO5) 1NT1, 1NT2, 1NT1 and HO5
Cases of F obtained from crosses with₁Generation (1NT1-F in the figure)₁Shown)
Is a result of detection of an allozyme marker.
In Experiment 1, by crossing 1NT1 and HO5
107F obtained₁All individuals have wild-type body color and GFP
It showed fluorescence. All of the individuals tested are
Both Zyme markers (PGM^aAnd PGM^b)
(FIG. 4).

3. Detection of GFP gene The presence of the GFP gene in nuclear transplants and their progeny was analyzed by PCR. The result is shown in FIG. The G
The FP gene was expressed in the seven nuclear transplants and the F
It was detected in all _one individuals (FIGS. 5A, B and C). Here, FIG. 5A shows the nuclear transplant and the F in Experiment 1.
₁ shows the presence of the GFP gene in _one generation. Here, in order from the left column, the plasmid, ER-1α-A
/ GFP-bearing donor, recipient, 1NT1, 1N
5 example of _{F 1} generation derived from T2,1NT1 (in FIG 1NT1F1
Is shown). FIG. 5B shows the presence of β-Act / GFP-N in the nuclear transplant in Experiment 2. The columns are, in order from the left, plasmid pβ-Act / GFP-N, donor, recipient, 2NT1-2NT5. FIG.
C indicates the presence of β-Act / GFP in _{F 1} generation, column starting from the left, plasmid, each 2NT1~2
Shows the _{F 1} generation for each three cases were obtained from a cross between NT5 and OR (shown as 2NT1F ₁ ~2NT5F ₁ is in the figure 5C).

The GFP gene was used in Experiment 1 and Experiment 2.
Expressing GFP in both ₂Detected in generations
(FIGS. 5D and E). Here, FIG.
F derived from 1₂ET-1α-A / GFP in generations
Indicates the presence of Columns are plasmid and firefly in order from the left.
F of 5 cases where light was observed₂Generation [ie, G in FIG. 5D
FP (+)], and 5 non-fluorescent Fs₂Generations [ie
GFP (-)] is shown in FIG. 5D.

FIG. 5E shows the F derived from 2NT1.₂
2 shows the presence of β-Act / GFP-N in generations. Mosquito
Lamb, in order from the left, plasmid and fluorescence were observed in 5 cases
F ₂Generation [ie, shown as GFP (+) in FIG. 5E
And 5 non-fluorescent Fs₂Generation [ie, G in FIG. 5E
FP (-)]. All endogenous EF-1α-A genes
Was amplified as a control in PCR analysis.

4. All 6 nuclear transplants (1NT1 and 2NT1-2NT) that reached the ploidy stage
5) was a diploid (6) having 48 chromosomes (Table 2).
All F ₁ generation examined derived from the nuclear transfer member was also diploid (data not shown).

[0066]

[Table 2]

5. Inheritance of Genetic Markers 1NT1 and 1NT2 showed wild type body color and GFP fluorescence. The melanophore differentiated in these individuals at a 2H embryo stage similar to that seen in normal development. 2NT1-2
NT5 all showed scarlet body color and GFP fluorescence (Table 2).

[0068] All 107 _{F 1} individuals obtained by crossing the 1NT1 and HO5 showed body color and GFP fluorescence of the wild-type. In addition, each nuclear transplant of 2NT1-2NT5 and OR
All fifty to one hundred fifteen _{F 1} individuals obtained from a cross between showed body color and GFP fluorescence scarlet.

[0069] For further analysis of the _{F 2} generation 1
It was crossed by five _{F 1} individuals from NT1 HO5 or OR paired matings (pair-mating). For _{F 2} generations 105-137 obtained in each mating were examined and their body color and GFP fluorescence at an embryonic stage of 6-day-old. The expression of the body color was 51.5% for wild type and scarlet, respectively.
± 3.4% and 48.5 ± 3.4% proportions, ie
Separation was at a ratio of 1: 1 (Table 3). Similarly, GFP fluorescence expression was positive and negative, 52.8 ± 3.2%, respectively.
And inherited at a rate of 47.3 ± 3.2%, ie, 1: 1 (Table 3). The body color and GFP fluorescence were inherited independently (data not shown).

In Experiment 2, 5 cells derived from each nuclear transplant were used.
Or _{F 1} individuals 6 crossed by OR paired mating to obtain the _{F 2} generation 103-163 from each mating. F tested
All _two generations showed scarlet body colors (Table 3). At the 6-day-old embryo stage, GFP positive and GFP negative individuals
From the percentages of 6.8 ± 4.7% and 53.3 ± 4.7% (progeny derived from 2NT4), 51.8 ± 3.1% and 4
It was inherited up to a percentage of 8.3 ± 3.1% (progeny derived from 2NT5), ie a 1: 1 ratio (Table 3).

[0071]

[Table 3]

6. Conclusion In conclusion, all these results from two series of experiments show that the nuclear transfer is diploid, fertile, and homozygous for the genetic marker of the donor nucleus, and These markers have been shown to be inherited in the next generation in a Mendelian fashion. The expression of the GFP gene in the nuclear transplant and their progeny are similar to those in the donor transgenic fish. The established nuclear transfer is an important step for cloning fish from somatic cells and cultured cells.

The totipotency of the nucleus of medaka blastula cells has been suggested by the transfer of germ cells to embryos resulting in germline chimerism [Y. Wakamatsu et al., Mol. Marine Biol. Biote.
ch. 2 (2), 325 (1983)], the results of this experiment showed that the nuclei of embryonic cells in the blastula stage of medaka are totipotent. In fish, cryopreservation of fertilized eggs is not possible due to the presence of large yolk mass, but cryopreservation of sperm is possible. The nuclear transfer can be used as a tool for the preservation of diploids in fish by combining the technique with cryopreservation of embryo cells.

The survival rate of nuclear transplants after the blastula stage was determined in Experiment 2.
Was lower in Experiment 1 than in. HNI-
I and OR are thought to belong to different populations from the northern and southern regions of Japan, respectively [M. Sakaizum
i, Genetica 69, 119 (1986)]. The contribution of genetic background to the efficiency of nuclear transfer has also recently been demonstrated in mice
(WMReideout III et al., Nature Genetics 24,109 (200
0)]. Genetic mismatch between the donor and recipient lines
It may be larger in Experiment 1 than in Experiment 2 where both donor and recipient fish are derived from OR.

All of the seven nuclear transplants (including 1NT2)
It was female, for unknown reasons.

The PGM allozyme marker of the recipient was not detected in 1NT1 and 1NT2. In addition, triploid individuals and individuals with only genetic markers of recipient fish were obtained by nuclear transfer into nucleated oocytes (Niwa, K. et al., (1999); Niwa, K . Et al., (2
000) supra], the individual was not obtained through the first and second experiments.

From these results, it was found that X-ray irradiation was effective for inactivating oocyte nuclei.

[0078]

The present invention provides a method for producing a cloned fish using embryo cells. Thereby, it is possible to produce the desired fish in large quantities. As a result, it is possible to enjoy benefits in many fields such as aquaculture, food, medicine, pet industry, and various test animal industries.

The present inventors have succeeded in producing, for the first time in the world, fertile fish clones using fish embryo cells. Embryonic cell cloning technology is the basis of cloning technology using somatic cells, and therefore the method provided by the present invention greatly contributes to the development of cloning technology for fish.

[Brief description of the drawings]

FIG. 1 is a schematic diagram illustrating the method of the present invention.

FIG. 2 is a photograph showing the morphology of an organism showing a killifish as an example of the recipient of the present invention.

FIG. 3. GF in donors, nuclear transplants and their progeny
The photograph which shows the form of the organism which shows the expression of a P gene.

FIG. 4 shows the results of detection of PGM Allozyme in nuclear transplant and F ₁ generation.

FIG. 5 is a view showing the results of detecting a GFP gene in a nuclear transplant and its progeny.

[Explanation of symbols]

1. Donor 2. Embryo 3. Embryo cells 4. Micropipette 5. Recipient 6. Oocyte
7. X-ray 8. Nuclear transfer

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA10 BA80 GA03 GA08 GA09 GA12 4B065 AA90X AA90Y AB04 AB10 AC20 BA04 BA08 BA30 BD50 CA60

Claims (3)

[Claims] 1. A method for producing a fish clone, comprising the steps of: irradiating a nucleus-receiving cell with X-rays; transplanting a nucleus of a nucleus-providing cell into the nucleus-receiving cell; And generating the nucleus-providing cell is an embryonic cell. 2. The method according to claim 1, wherein the nuclear receptor cells are oocytes. 3. A cloned fish produced using the method according to claim 1 or 2.