JPH104975A - Improvement of meat quality and animal improved in meat quality - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the meat quality of a non-human animal by transducing a recombinant DNA into the chromosomal DNA of the productive cell, germinal cell, etc., of the non-human animal, wherein the gene of a protein, etc., constituting the flavor, taste, nutrient, etc., of the meat is lighted to the gene control region of the recombined DNA. SOLUTION: This method for improving the meat quality of a non-human animal comprises ligating a DNA coding a protein (e.g. myoglobine) relating to an expression of the flavor, taste, nutrient, etc., of the meat or a protein related to the expression of the flavor, the texture, the nutrient, etc., to the gene control region of a gene related to the production of the protein, and subsequently transducing the recombinant DNA into either of the fertilized egg, productive cell and germinal cell of the non-human animal to transform the non-human animal cell, or transducing the recombinant DNA into the protein production-related gene region of the chromosomal DNA of either of the fertilized egg, productive cell and germinal cell of the non-human animal by a homologous recombination method to transducer the non-human animal cell. Thus, the meat quality of the non-human animal is improved by the gene manipulation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a technique for improving meat quality of meat, and more particularly, to a method for improving meat quality by genetic manipulation and an animal having improved meat quality by genetic manipulation.

[0002]

2. Description of the Related Art Conventionally, humans have been
He has eaten animal meat vigorously. In particular, beef tends to be eaten more than other meat and fish such as pork and chicken.
As a result, there are currently about 1.3 billion cattle on this planet. These cows eat about 25% of the world's land area and eat enough grain to feed hundreds of millions of people. Thus, adult cows are about ten times as heavy as humans, so their combined weight is much higher than the total weight of all humans on earth.

[0003]

However, the growing number of cattle disrupts the Earth's ecosystem and destroys the habitats of continents. Cattle breeding is a major cause of global rainforest destruction. In Central and South America, vast virgin forests are being cleared to create cattle ranches. Desertification, which is occurring in the southern margins of the Sahara Desert in Africa and pastures in the western United States and western Australia, is primarily due to cattle grazing. Overgrazing in the arid / semi-arid regions of the continents has created a dry, barren desert. The bovine excreta overflowing from the breeding ground
It is a major source of organic pollution in groundwater. Cattle are also a source of methane, one of the major greenhouse gases that acts to trap heat in the atmosphere.

[0004] As described above, the environment is destroyed by breeding cows having flavors that match the tastes of human beings using conventional techniques. In addition, cattle breeding is also the most expensive in livestock breeding. Therefore, the present invention
An object of the present invention is to produce a non-human animal having meat having a flavor that matches human taste by genetic manipulation and to improve the meat quality of the non-human animal.

[0005]

Means for Solving the Problems The first invention is a method for improving the meat quality of a non-human animal, comprising a protein constituting meat flavor, texture, nutrients, etc. or its flavor, texture, and the like. DNA encoding a protein involved in the expression of nutrients and the like (hereinafter referred to as meat quality-related protein)
Is introduced into a fertilized egg, germ cell, or embryo cell of a non-human animal to transform the non-human animal. According to the present invention, when a meat-related protein is expressed in a non-human animal,
The meat quality of the non-human animal is improved. In this specification, the protein involved in the expression of meat flavor, texture, nutrients, and the like include, for example, amino acids, nucleic acids, lipids, or enzymes or enzyme groups that synthesize certain proteins that constitute the flavor, It means a functional protein or a group of functional proteins.

[0006] The second invention is a method for improving the meat quality of a non-human animal, comprising a DN encoding a meat quality-related protein.
A is introduced into a fertilized egg, germ cell or embryo cell of a non-human animal, and the non-human animal is transformed by introducing a recombinant DNA obtained by binding A to a gene regulatory region of a gene involved in the production of the protein. A method characterized by the following. According to the present invention, a DN encoding a meat quality-related protein
Since A is linked to the gene regulatory region of the gene involved in the protein production to form a recombinant DNA, the meat-related protein is expressed in the non-human animal according to the expression mode of the meat-related protein. .

[0007] A third invention is a method for improving the meat quality of a non-human animal, which comprises a DN encoding a meat quality-related protein.
A is a recombinant gene obtained by binding A to a gene regulatory region of a gene involved in protein production, and a gene involved in protein production of any chromosomal DNA of a fertilized egg, germ cell, or embryo cell of a non-human animal And transforming the non-human animal by homologous recombination. According to the present invention, a recombinant DNA formed by binding a DNA encoding a meat quality-related protein to a gene regulatory region of a gene involved in the production of the protein can be used for production of a protein of a non-human animal by homologous recombination. Since the substitution is introduced into the region of the gene involved, the meat-related protein is expressed in a non-human animal and the protein encoded by the substituted region is not expressed due to the expression mode of the meat-related protein.

[0008] A fourth aspect of the present invention is a method for improving the meat quality of a non-human animal, the method comprising a DN encoding a meat quality-related protein.
The recombinant DNA containing A is placed on the downstream side of the gene regulatory region of a gene involved in protein production, in any of the chromosomal DNA of a fertilized egg, germ cell, or embryo cell of a non-human animal,
A method for improving the meat quality of a non-human animal, characterized in that the non-human animal is transformed by introduction by homologous recombination. According to the present invention, a recombinant DNA containing a DNA encoding a meat quality-related protein is obtained by homologous recombination.
Since the gene regulatory region of the gene involved in the production of the protein of the non-human animal has been replaced and introduced, the meat quality-related protein is expressed by the expression pattern of the protein of the non-human animal. At the same time, the protein encoded in the replaced region is not expressed.

A fifth aspect of the present invention is a non-human animal, characterized by being transformed by introduction of a DNA encoding a meat quality-related protein. According to the present invention, the meat quality-related protein is expressed in the non-human animal, so that the meat quality is improved in the non-human animal.

[0010]

Embodiments of the present invention will be described below. (Flavor, texture, nutrients, etc. of meat) Flavor, texture, nutrients, etc. of meat (hereinafter, simply referred to as flavor) are proteins, lipids, sugars, nucleic acids, and amino acids contained in meat or blood. They can be used alone or in combination. Therefore, in the present invention, the meat-related protein, that is, the protein involved in the expression of the meat flavor, directly, in addition to the protein constituting the meat flavor, lipids, sugars, nucleic acids, constituting the meat flavor Proteins (enzymes) that indirectly make up the flavor of meat by synthesizing amino acids
Is also included.

The unique flavor of meat is attributed to the protein itself in meat or blood, or to the aforementioned compounds such as lipids, saccharides, nucleic acids, and amino acids synthesized by proteins (enzymes), or to the meat. This is because the composition of the protein or compound is different from that of other animals. Therefore, by expressing proteins or other compounds that constitute meat flavors that match human tastes in non-human animals that do not destroy the natural environment at low cost, these non-human animals can be used as new livestock animals. can do.

Here, proteins that directly or indirectly constitute the flavor of meat include myoglobin, hemoglobin, collagen, actin, myosin, troponin, titin, nebulin, tropomyosin, actomyosin, α-actinin, β -Actinin, Z protein, phosphothrylase, connectin, nebulin, creatine kinase, lactate dehydrogenase, parvalbumin, carbaine, transglutaminase, calmodulin,
Elastin, fibrinogen, globulin, reticulin and the like can be mentioned.

[0013] The lipids constituting the meat flavor include:
Examples include neutral fats such as monoglycerides, diglycerides, and triglycerides, phospholipids, glycolipids, and cholesterol. Fatty acids constituting monoglycerides, diglycerides, and triglycerides include myristic acid, myristooleic acid, palmitic acid, palmitooleic acid, stearic acid, oleic acid, linoleic acid,
Linolenic acid, oleic acid, palmitooleic acid, linoleic acid, linolenic acid and the like can be mentioned.

The phospholipids include phosphatidic acid, phosphatidylglycerin, cardiolipin,
Examples include phosphatidylcholine, phosphatidylinositol, phosphatidylethanol, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositolamine, ether glycerophospholipid, lysophospholipid, sphingomyelin and the like.

[0015] Examples of glycolipids include cerebroside, galactoside, sulfolipid, and mucolipid.

(Non-human animal) In the present invention, the non-human animal refers to an animal other than a human, in addition to mammals, reptiles,
Includes amphibians, birds, and fish. Preferred non-human animals in the present invention are mammals and birds for which domestic breeding technology has been established, and fishes for which aquaculture technology or release technology has been established. As such mammals, specifically, cattle,
Major livestock animals such as pigs, horses and sheep can be mentioned.
Also, such birds include chicken, duck, quail, turkey,
Pigeons and the like can be mentioned. Examples of such fish include hamachi, eel, Thailand, loach, salmon, trout, and sweetfish.

The meat quality-related protein may be derived from an animal of a different species from the non-human animal in which the protein is to be expressed, or may be derived from an animal of the same species.
Further, it may be an artificial protein.

DNA encoding a meat quality-related protein
Is incorporated into, for example, a general expression vector. By introducing a gene using this expression vector, a DNA encoding a meat quality-related protein is inserted and added to a chromosomal DNA of a non-human animal together with an expression promoter and the like by random recombination. As a result, the protein of another non-human animal is expressed in the non-human animal.

Further, D which encodes a meat quality-related protein
NA is inserted and added to chromosomal DNA of a non-human animal by random recombination as a recombinant DNA linked to a gene regulatory region of a gene involved in protein production.

Further, a recombinant DNA containing a DNA encoding a meat quality-related protein is formed so as to be capable of homologous recombination downstream of a gene regulatory region of a gene involved in the production of a protein of a non-human animal. By replacement, it is inserted into chromosomal DNA of a non-human animal. This recombinant DNA is
Having a region encoding a meat quality-related protein,
The coding region is sandwiched between the upstream and downstream sides of the coding region, and has a homologous region with a gene involved in the production of a protein of a nonhuman animal to be introduced. By this gene transfer, the gene located downstream of the gene regulatory region in the non-human animal is destroyed and cannot be expressed, while the newly inserted DNA expresses the meat quality-related protein by the expression mode of the gene.

Further, D encoding a meat quality-related protein
Recombinant DNA in which NΛ is linked to the downstream of a gene regulatory region of a gene involved in the production of a meat quality-related protein.
Is formed to be capable of homologous recombination with a region of a gene involved in the production of a protein of a non-human animal, and by homologous recombination,
Insert into chromosomal DNA of non-human animal. This recombinant DN
A has a region encoding a meat quality-related protein, and a gene regulatory region of a gene involved in producing the meat quality-related protein upstream of the code, and has a region upstream of these regions sandwiching these regions. It has a region homologous to a gene involved in the production of a protein of a non-human animal to be introduced downstream. By this gene transfer, the meat-related protein is expressed in the non-human animal according to the expression mode of the meat-related protein. on the other hand,
Non-human animal proteins are not produced because the genes involved in the production of non-human animal proteins are disrupted.

(Gene introduction) Such a recombinant DNA
Is introduced into fertilized eggs, germ cells, and embryo cells of non-human animals. In the case of introduction, a microinjection method, a synthetic lipid method, an electroporation method, or a direct injection method into a reproductive organ is used.

(Detection) To determine whether or not the recombinant DNA has been introduced, a part of the obtained offspring is collected, DNA is extracted, and PC is detected using a primer capable of detecting a foreign gene.
The amplification is performed by the R method. Alternatively, whether or not a desired protein is expressed by the introduced DNA can be determined by a known method such as an RTPCR method or a method of directly detecting a protein.

(Transgenic animal) In this way, the transgenic animal is transformed by introducing the recombinant DNA, and the introduced DNA is also introduced into the germ cells, whereby the trait due to the foreign DNA is transmitted. By crossing these individuals, progeny that are homozygous for the foreign DNA can be obtained.

[0025]

According to the method for improving meat quality and the transformed animal, the meat quality-related protein is expressed in the non-human animal, and the meat quality of the non-human animal is improved. Further, by introducing a DNA encoding a meat quality-related protein by homologous recombination, a meat quality-related protein is expressed instead of a protein expressed in a non-human animal. As a result, the meat quality of the non-human animal is effectively improved.

[0026]

The following describes an experimental example in which the bovine myoglobin gene was introduced into a pig cell line PK15 for transformation. (Extraction of bovine myoglobin cDNA) First, 0.1 g of bovine muscle was collected and collected from Maniatis, T .; Et al. (Maniatis, T. et al Molecular).
ar Cloning, 2nded. , Cold Sp
ring Harbor Laboratory, Co
ld SpringHarbor. New York
7.3-7.36, 1989). For the extracted RNA, primer BT-45 '
-CTGTCGGAGACAGACACCCAGTCA
-3 'and primer BT-994 5'-GTTGC
RTGA was performed using AGACACCCATGCCCTCCATGC-3 ′, and bovine myoglobin cDNA was
(Hereinafter, simply referred to as myocDNA).

(Preparation of Transformation Construct) The obtained myo cDNA was cloned into PCR3 (inv
5'-CAATTTACGCGCGCGC as a primer for the upstream portion of the promoter after insertion into the designated insertion site
Using GTTTGACATTGA-3 ′, 5′-GTTTATCCCC as a primer for the downstream portion of polyA
Using TGATTCCGCGGATAAC-3 ', P
By performing the CR, p from the start of the promoter
The region up to the end of the oligoA signal was extracted, and this D
The NA fragment was used as an introduction construct.

(Transformation of Pig Cell Line PK15) PK
The above-described construct for introduction was introduced into 15 cells using Lipofectin. On the first day, solution A (mixture of 1 μl of the construct solution for introduction and 9 μl of DMEM)
Was mixed with solution B (mixture of 5 μl of lipofectin and 5 μl of DMEM), mixed gently, and left at room temperature for 15 minutes.
Then, this mixed solution was added to a culture dish of PK15 cells in the course of growth, and the mixture was gently stirred. On the second day, the medium was
Exchanged for YMI and on day 3 selected drug G418 was given for 20
Add 0 μl / medium. On the seventh day, the medium was changed to RPM.
After replacing with I, the supernatant was discarded, and a colony obtained one week later was collected.

(Confirmation of introduction of myocDNA) Cells were collected from the obtained colonies and added to a DNA extraction solution.
DNA was extracted. PCR was performed on the extracted DNA using the primers BT-4 and BT-994. The PCR solution was 77 μl of water, 10 μM 10 μM
1, dNTP 8 μl, BT-42 μl, BT-994
2 μl, Taq Polymerase 0.5 μl,
1 μl of DNA was used. 10% of the solution after PCR was subjected to 1.2% agarose gel electrophoresis,
A band derived from bovine myoglobin DNA was confirmed at around 0 bp. As a result, it was confirmed that bovine myoglobin was introduced into the PK15 cells.

Claims (5)

[Claims] 1. A method for improving meat quality of a non-human animal, comprising: a protein constituting meat flavor, texture, nutrients, etc. or a DNA encoding a protein involved in expression of the flavor, texture, nutrients, etc. A method comprising introducing a recombinant DNA containing the compound into a fertilized egg, germ cell, or embryo cell of a non-human animal to transform the non-human animal. 2. A method for improving meat quality of a non-human animal, comprising: a protein constituting meat flavor, texture, nutrients, etc., or a DNA encoding a protein involved in the expression of the flavor, texture, nutrients, etc. DNA linked to a gene regulatory region of a gene involved in protein production
Is introduced into any of fertilized eggs, germ cells, and embryo cells of a non-human animal to transform the non-human animal. 3. A method for improving meat quality of a non-human animal, comprising a protein constituting meat flavor, texture, nutrients, etc. or a DNA encoding a protein involved in expression of the flavor, texture, nutrients, etc. DNA linked to a gene regulatory region of a gene involved in protein production
To transform a non-human animal by homologous recombination into a region of a gene involved in protein production in chromosomal DNA of any of fertilized eggs, germ cells, and embryo cells of the non-human animal. Features method. 4. A method for improving meat quality of a non-human animal, comprising a protein constituting meat flavor, texture, nutrients, etc. or a DNA encoding a protein involved in expression of the flavor, texture, nutrients, etc. The recombinant DNA containing the non-human animal fertilized eggs, germ cells, germ cells of any chromosome D
A method for improving the meat quality of a non-human animal, comprising introducing the NA into the downstream of a gene regulatory region of a gene involved in protein production by homologous recombination to transform the non-human animal. 5. A non-human animal, which is transformed by introducing a DNA encoding a protein constituting meat flavor, texture, nutrients, etc. or a protein involved in expression of the flavor, texture, nutrients, etc. A non-human animal, which is characterized in that: