WO2004066724A1 - 脂肪酸不飽和化酵素を有する形質転換動物およびその作製方法 - Google Patents
脂肪酸不飽和化酵素を有する形質転換動物およびその作製方法 Download PDFInfo
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- WO2004066724A1 WO2004066724A1 PCT/JP2003/000930 JP0300930W WO2004066724A1 WO 2004066724 A1 WO2004066724 A1 WO 2004066724A1 JP 0300930 W JP0300930 W JP 0300930W WO 2004066724 A1 WO2004066724 A1 WO 2004066724A1
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- Prior art keywords
- fatty acid
- animal
- desaturase gene
- acid desaturase
- gene
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
Definitions
- the present invention relates to a transformed animal into which a gene for a fatty acid desaturase has been introduced, a transformed animal cell, and a method for increasing the unsaturated fatty acid content in an animal.
- Fat is a very important source of energy, along with essential fatty acids (linoleic acid (18: 2n-6), a-linoleic acid (18: It is also a source of 3n_3) and arachidonic acid (18: 4 ⁇ -3)).
- essential fatty acids lactioleic acid (18: 2n-6), a-linoleic acid (18: It is also a source of 3n_3) and arachidonic acid (18: 4 ⁇ -3).
- Crawford et al. Am J Clin Nutr, 31, 2181-2185, 1978
- ⁇ 12 fatty acid desaturase and ⁇ 15 fatty acid desaturase are present in plants and can synthesize these polyunsaturated fatty acids.
- linoleic acid, polylinolenic acid, and EPA contains a large amount of polyunsaturated fatty acids such as DHA and DHA.
- ⁇ 12 fatty acid By desaturase is meant an enzyme that catalyzes a reaction that creates a double bond at the 12th position from the terminal lipoxyl group in the fatty acid carbon chain.
- a ⁇ 15 fatty acid desaturase refers to an enzyme that catalyzes a reaction to form a double bond at the 15-position position from the terminal lipoxyl group in the fatty acid carbon chain.
- ingested linoleic acid can be converted to arachidonic acid (20: 4n-3 / n-6), and ⁇ -linolenic acid can be converted to EPA or DHA.
- Chemical formula 1 shows the structure of palmitic acid (having 16 carbon atoms and no unsaturated bond, 16: 0), and chemical formula 2 shows the structure of stearic acid (having 18 carbon atoms and no unsaturated bond, 18: 0). Fatty acid having 18 carbon atoms and having a double bond between the 9th and 10th carbon atoms counted from the carboxyl group is oleic acid.
- Chemical formula 3 shows oleic acid (unsaturated bond with 18 carbon atoms) One is 18: 1).
- the fatty acid having a double bond at the 12-position is linoleic acid
- the chemical formula 4 shows linoleic acid (18 unsaturated bonds with 18 carbon atoms, 18: 2) The structure of is shown.
- a fatty acid having a double bond at the 15-position is linolenic acid.
- Chemical formula 5 shows ⁇ -linolenic acid (18 unsaturated bonds with 18 carbon atoms, 18: 3) The structure of is shown.
- a transgenic animal wherein the unsaturated fatty acid content in the animal is increased by transforming the animal with the saturase gene and expressing the fatty acid desaturase gene in the animal.
- a second aspect of the present invention is to transform an animal cell with a fatty acid desaturase gene and express the fatty acid desaturase gene in the animal cell, thereby increasing the unsaturated fatty acid content in the animal cell.
- a transformed animal cell is to transform an animal cell with a fatty acid desaturase gene and express the fatty acid desaturase gene in the animal cell, thereby increasing the unsaturated fatty acid content in the animal cell.
- a third aspect of the present invention relates to an animal comprising transforming an animal with a fatty acid desaturase gene and expressing the fatty acid desaturase gene in the animal. In this method, the unsaturated fatty acid content is increased.
- a fourth aspect of the present invention is to construct a vector, which is an expression plasmid having a transcriptional regulatory region for promoting the expression of a fatty acid desaturase gene and the fatty acid desaturase gene, and Transformation is performed by introducing the cells into early embryos, somatic cells, and embryonic stem cells (ES cells) of an animal, and an individual animal is generated from the transformed early embryo, somatic cells, and embryonic stem cells of the animal.
- ES cells embryonic stem cells
- a method for increasing the unsaturated fatty acid content in an animal comprising the step of expressing the fatty acid unsaturated enzyme gene in an animal individual.
- FIG. 1 is a photograph of RT-PCR analysis and Northern blot analysis showing the expression of spinach fad2 gene in transgenic mice.
- FIG. 2 is a photograph of Western blot analysis showing the expression of spinach FAD2 in brown adipose tissue of a transgenic mouse.
- FIG. 3 is a graph showing the fatty acid composition ratio of total fat in brown adipose tissue between wild type mice and B1 strain mice to which a normal diet or an oleic acid diet was administered.
- Figure 4 is a photograph of Southern blot analysis showing the integration of the transgene into the chromosome in transgenic plants and their offspring.
- FIG. 5 is a photograph of RT-PCR analysis showing fad2niRNA expression in white adipose tissue of transgenic plants (B3, B12 and B19).
- FIG. 6 is a photograph of Northern blot analysis showing the expression of fad2 mRNA in a B-12 strain-transgenic pig.
- Figure 7 shows white fat groups of aP2 / fad2-introduced bush (B-12 strain) reared on a normal diet. It is a graph which shows the fatty acid composition ratio in the total lipid of a weave. Detailed description of preferred embodiments
- the present inventors isolated an endoplasmic reticulum membrane-bound fatty acid desaturase gene obtained from a plant, injected it into fertilized eggs of mice and pigs, stably introduced it on its chromosome, and developed it into individuals. I let it. When these individuals were examined in detail, the fatty acid composition was altered by the expression and function of the introduced fatty acid desaturase gene without affecting the normal development and function of the animals, and the polyvalent polyunsaturation was observed. It was found that the saturated fatty acid content increased.
- the present invention provides an animal individual and an animal cell into which a fatty acid desaturase gene is introduced, expressed and functions, and a method for producing the same.
- Preferred fatty acid desaturase genes used in the practice of the present invention include fatty acid desaturase genes that are present in plants but are not endogenous in animals. More specific examples thereof include a ⁇ 12 fatty acid desaturase gene and a ⁇ 15 fatty acid desaturase gene.
- the scope of the present invention is not limited thereto, and other fatty acid desaturases can be used as long as they are useful for increasing the expression level of fatty acid desaturase and function in animals. It is also possible to use genes or genes that increase the level of unsaturated fatty acids.
- animals lack ⁇ 12 and 1515 fatty acid desaturases, so they cannot biosynthesize linoleic acid or a-linolenic acid. Need to take. Therefore, by expressing plant-derived ⁇ 12 fatty acid desaturase and ⁇ 15 fatty acid desaturase in animals, it becomes possible to increase the content of unsaturated fatty acids that are useful for health in animals. In order to achieve the desired effects in the present invention, it is possible to express only one of the ⁇ 12 fatty acid desaturase and the ⁇ 15 fatty acid desaturase, or to express both of them. It is also possible.
- ⁇ 12 fatty acid desaturase gene derived from spinach root used in the following Examples.
- Delta 1 fatty acid desaturases are known to have an endoplasmic reticulum membrane-bound form present in the endoplasmic reticulum membrane and a chloroplast form present in the chloroplast membrane.
- the electron transport system of ferredoxin which is unique to plants, is used.
- the enzyme derived from the root of spinach is an endoplasmic reticulum membrane-bound enzyme and performs desaturation using the cytochrome b electron transport system that is also present in animals. Since the present invention expresses fatty acid desaturase in animals, it is preferable to use a gene encoding an endoplasmic reticulum membrane-bound enzyme that can be easily used by animals.
- fatty acid desaturase gene that can be used in the present invention is not necessarily limited to a plant-derived gene. Traditionally, animals were considered to have no ⁇ 12 and 15 fatty acid desaturases. But in recent years, nematodes
- ⁇ 12 fatty acid desaturase and ⁇ 15 fatty acid desaturase were isolated from (C. elegans).
- Such animal-derived enzymes can also be used as long as they are useful for the purpose of the present invention of increasing the content of unsaturated fatty acids that are useful for health, and such embodiments are also considered to be within the scope of the present invention. It should be.
- the animals used in the present invention include any animals used for food.
- Food animals which are mammals, are particularly preferred as animals used for the purpose of the present invention because of their generally low unsaturated fatty acid content and relatively easy transformation.
- bush, sea lion, goat, sheep and the like are particularly preferable target animals for transformation.
- birds such as chickens and quails, and fish such as tuna are also target animals for transformation.
- the scope of the transgenic animals of the present invention It is not limited and contains a wide range of animals used for food.
- not only the transformed animal individuals but also animal cells into which the fatty acid desaturase has been introduced are within the scope of the present invention.
- a transcription regulatory region for promoting the expression of a fatty acid desaturase gene is ligated upstream of the gene for the fatty acid desaturase, thereby constructing an expression plasmid, Bekuyuichi.
- the gene of the fatty acid desaturase may be cDNA encoding the enzyme or genomic DNA.
- Preferred promoters to be used herein include the promoter region of the yeast ⁇ -actin and the adipocyteP2 (aP2) promoter used in the following Examples, and further, the UCP promoter, SV40 promoter, and cytomegavirus. Examples include, but are not limited to, promoters and the like, and various promoters generally used in this technical field can be used. These promoters can be appropriately selected by those skilled in the art according to the target expression site and expression time. For the purpose of systemic expression, it is preferable to select the i6-actin promoter.For the purpose of specific expression in adipose tissue, the expression is specifically regulated in white adipose tissue and brown adipose tissue. An aP2 promoter that performs the following would be preferred.
- the expression plasmid is also not particularly limited, and various plasmids generally used in this technical field can be used.
- PUC118 and pBluescript I IS used in the following examples are particularly preferred, but PSV2,; 3 ⁇ , pZip, C012, ME18S and the like can also be used.
- Transformation is performed by introducing the vector thus prepared into an early embryo, somatic cell, or embryonic stem cell (ES cell) of an animal, and transforming the early embryo, somatic cell, embryonic stem cell of the animal. Generate more animal individuals.
- the constructed vector is introduced into an early embryo, but it is also possible to use a cloning technique and a chimera formation method to generate an individual after introduction into animal cells such as somatic cells and ES cells. is there.
- a gene may be introduced into an arbitrary or specific tissue or organ of a living body by various methods, and the introduced gene may function in the tissue or organ so that the enzyme biosynthesizes unsaturated fatty acids. It is also possible.
- the purpose of this study was to examine whether plant-derived endoplasmic reticulum membrane-bound fatty acid desaturase gene functions in animal animals to convert fatty acids contained in individual fats into unsaturated fatty acids.
- a 1.5 kbp linear fragment containing the chicken ⁇ -actin promoter region was subcloned with SG5 1.7
- the SV40 splicing region and the sv40 poly (A) additional signal region are located upstream and downstream of the cDNA fragment (Hindlll-EcoRI) of ⁇ 12 fatty acid desaturase (hereinafter referred to as fad2) isolated from the root of spinach. Ligation and insertion into the PUC118 vector.
- a 4.3 kbp fragment (PstI-BamHI, / 3-act / fad2) of the constructed plasmid was electrophoresed on a 1% agarose gel, purified with Geneciean II (BIO 101 Inc.), and purified with TE buffer (PH7.4). Dissolved in zg / ml. The gene thus constructed was introduced into a mouse.
- mice were purchased from Kiwa Experimental Animals Co., Ltd. The handling of these animals was performed according to “Guidelines on Animal Experiments” (edited by the Japan Society of Experimental Animal Science, Soft Science Inc., 1991). Microinjection of DNA into the pronucleus of the mouse fertilized egg and pseudopregnancy of the surviving embryo Transfer to the oviduct of the mouse is performed by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, 157-173, New York: Cold Spring Harbor Laboratory) Press, 1986). When 3] -act / f ad2 was microinjected into 1219 fertilized eggs and transplanted into fertilized females, 111 offspring were obtained.
- NT represents a wild-type mouse
- B1 represents a transgenic mouse of the B1 strain
- B2 represents a transgenic mouse of the B2 strain
- B6 represents a transgenic mouse of the B6 strain.
- FIG. 1A shows the results of RT-PCR analysis of iad2 mRNA in three lines of transgenic mice (Bl, B2 and B6).
- RT-PCR analysis showed that each mouse tissue (brain, heart, lung, liver, Spleen, kidney, skeletal muscle, ovary, testis and white fat, brown fat) were collected and total RM was extracted with Trizol (Gibco / BRL).
- Br is brain
- 3 ⁇ 4 is heart
- Lu is lung
- Li Li
- Sp spleen
- Ki kidney
- SM skeletal muscle
- 0v ovary
- Te testis
- BA brown adipose tissue
- WA white Adipose tissue is shown.
- Arrows indicate fad2-specific transcripts (378 bps).
- G3PDH is a positive control.
- RNAs were reverse transcribed using AMV reverse transcriptase and random primers (Yukara) to obtain cDNA.
- cDNAs were amplified by PCR using fad2 specific primers (5'-CTCTCCAATCTACTCGGAC-3 'and 5'-ATTGGCTTTATAGCCTTGGT-3'). Amplification products were electrophoresed in a 2% agarose gel. As a control, amplification using a specific primer of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene (Clontech) was also performed.
- G3PDH glyceraldehyde-3-phosphate dehydrogenase
- 1A shows the results of analysis of fad2 mRNA by RT-PCR in three transgenic mice of Bl, B2 and B6. As a result, expression at the mRNA level was constitutively confirmed in all tissues in mice of the Bl, B2 and B6 strains.
- FIG. 1B shows the results of Northern blot analysis of fad2 mRNA in the three lines of transgenic mice (Bl, B2 and B6).
- Br indicates brain
- WA indicates white adipose tissue
- BA indicates brown adipose tissue
- Li indicates liver.
- Arrows indicate fad2-specific transcripts (1.4 kbp).
- -act in is a positive control.
- Total RNA was extracted with Trizol from the tissues of the brain, white fat, brown fat, and liver of mice of the Bl, B2, and B6 strains whose expression was confirmed by RT-PCR analysis.
- poly (A) RNA was further purified using Oligotex-dT (Yukara), electrophoresed with 1% agarose-formaldehyde, and blotted on a nylon membrane (Amersham) with 20 ⁇ SSC.
- Oligotex-dT Yukara
- the blotted membrane was hybridized with a probe of spinach-derived FAD2 cDNA (1.7 kbp) labeled with 32 P-dCTP by a random primer method and a mouse s-actin probe as a control.
- the expression level of the iad2 gene was different between tissues and was highly expressed in brain and brown adipose tissue (Fig. 1B). Such a difference in expression level between tissues was confirmed by other reports using chicken s-actin as a promoter (Matsumoto et al., Mol Reprod Dev, 39-136-140). (1994), Mat sumo to et al., Mem Res Inst BOST, Kinki Univ, 2, 11-18 (1999)). This indicates that the introduced fatty acid desaturase gene is strongly transcribed in each mouse tissue, particularly in brown adipose tissue. The expression level was higher in the B1 and B2 lines than in the B6 line, and the B2 line had lower fertility, so the B1 line was used for further analysis. .
- proteins were extracted from the brown adipose tissue of the B1 line, which had the strongest expression at the mRNA level, and subjected to Western blot analysis.
- the collected brown adipose tissue was disrupted using a polytron homogenizer in a sucrose-TM buffer containing proteazein hippiter (Co-lete, Boehlinger Manheim).
- the suspension was centrifuged at 10000 g at 4 ° C for 10 minutes, and the supernatant was further centrifuged at 4 and 5000 g for 60 minutes to obtain a microsomal fraction.
- the sediment was resuspended in sucrose-TM buffer and the protein concentration was measured.
- a 50 / g protein equivalent was electrophoresed on a 10% SDS polyacrylamide gel and then transferred to a PVDF membrane (Millipore).
- the membrane was incubated for 1 hour with PROC-ACE (Dainippon Pharmaceutical) containing 0.2% Tween 20.
- the membrane was washed three times with PBS containing 0.5% Tween 20 (pH 7.2), and then incubated with Egret anti-FAD2 antiserum (1: 2,500) for 1 hour at room temperature.
- This antiserum was obtained by administering a synthetic oligonucleotide corresponding to 18 amino acid residues near the C-terminus of oleic acid desaturase from spinach to rabbits.
- the membrane was then washed with PBST, incubated for 30 minutes at room temperature with anti-Egret enzyme-labeled secondary antibody (1: 5000, Amersham) from the mouth, washed with TBTS, and detected by chemiluminescence (ECL, Amersham).
- FIG. 2 shows the results.
- Cont is a positive control and indicates the protein extracted from the spinach root.
- NT is a negative control and indicates the protein extracted from wild-type mice.
- B1 is a transgenic mouse of B1 strain
- B2 is It is a transgenic mouse of the B2 strain.
- Arrow indicates FAD2 (45 kDa).
- a protein of about 45 kDa which is the expected molecular weight of spinach FAD2
- mice Diets administered to mice included normal diet (MF, Oriental yeast, Table 1), non-fat diet (0.7% fat, Oriental yeast, Tables 2 and 3) and 5% oleic acid (01008, Sigma) Oleic acid feed (Oriental yeast, Table 4) was administered. All diets had the same calorie as 340kCal / 100g. Table 1 Component values of normal mice
- Wild-type and transgenic mice grown on normal diet were used. Wild-type mice were bred on a normal diet, fat-free diet and oleic acid diet, and transgenic mice were bred on an oleic acid diet for one week. After breeding, brown adipose tissue was collected and fatty acid composition was examined. The lipids in the brown adipose tissue were extracted with black-mouth-methanol (BUgh and Dyer, Can J Biochem Physiol, 37, 911-917 (1959)), and the fatty acid composition in the lipids was analyzed by gas chromatography (Wada and Murata, Plant Cell Physiol, 30, 971-978 (1989)) 0
- Figure 3 shows the fatty acid composition ratios in the total lipids of the brown adipose tissue of wild-type mice and B1 strain transgenic mice administered a non-fat diet or oleic acid diet for 2 weeks after growth on a normal diet.
- the vertical bar indicates the average value
- the error bar indicates the standard error.
- Statistical analysis was performed by Fisher's PLSD test after analysis of variance. As a result of examining the fatty acid composition of the brown adipose tissue, as shown in Fig.
- the ratio (Mol%) of linoleic acid (18: 2 ⁇ -6) in the total fatty acids of the B1 strain mice reared on an oleic acid diet was 1.6 and 1.4 times, respectively, of wild-type mice reared on a oleic acid diet (0.05).
- the oleic acid (18: 1 ⁇ -6) ratio decreased, and was at the same level as that of wild-type mice fed on a non-fat diet.
- the ratio of fatty acids in transgenic mice reared on an oleic acid diet was at the same level as in wild-type mice reared on a non-fat diet, except for linoleic acid.
- oleic acid (18: 1) serving as a substrate was added to the diet and bred for a certain period. It is known that increasing oleic acid content in the diet increases oleic acid content in adipose tissue (Klingenberg et al., Co. Biochem Physiol, HOB, 183-192, 1995). Because it is. Immediately before and 8 weeks after administration of the oleic acid diet, adipose tissue was collected to examine fatty acid composition.
- the transgene was constructed to express the fatty acid desaturase gene specifically in adipocytes of pig living bodies.
- a cDNA fragment (1.7 kb) of a ⁇ 12 fatty acid desaturase isolated from spinach root was cloned into vector pBluescript IIKS.
- a mouse adipocyte P2 (aP2) promoter region (5 kb) for inducing gene expression specifically in adipocytes was ligated upstream of this cDNA, and an SV40 splicing region and an SV40 poly (A) addition signal region were ligated downstream.
- the constructed plasmid was digested with restriction enzymes SacII and Xhol.
- the cut DNA was subjected to 1% agarose gel electrophoresis, and a DNA fragment of the transgene (about 7.5 kb) was isolated.
- the isolated transgene was purified by Geneclean II (BIO 101 Inc.) and then dissolved in TE buffer (pH 7.4) at 4/2 g / ml. The gene constructed in this way was introduced into Busu.
- the fusion gene was microinjected into the porcine early embryo pronucleus. Animals were handled according to the “Guidelines on Animal Experiments” (edited by the Japan Society of Experimental Animal Science, Soft Science Corporation, 1991). Sampling of embryos • Gene injection • Embryo transfer was performed as follows. Approximately 13 months old pigs (Durok () X F1; Landrace X Large White) weighing about 100 kg) were intramuscularly administered eCGlOOO IU, and 500 hours later hCG was administered 72 hours later. Pigs that exhibited estrus 24 hours after hCG administration were mated with male pigs.
- FIG. 4 shows the results of Southern blot analysis of the integration of the transgene into the chromosome in the transgenic pig. The results of analysis of litters of transgenic plants are also shown, which will be described in detail later. In Fig.
- B-12 is a transgenic pig () produced by injecting the aP2 / fad2 gene into the early embryo.
- the F1 litter was bred with B-12.
- the arrow indicates the fad2 gene, and integration of the transgene into the chromosome was observed in the transfected B-12, which was isolated from the transgenic spinach root tissue. It shows that the fad2 gene is stably integrated on the chromosome of bushus, but two of these six bushus were stillborn and one was postpartum-killed.
- Figure 5 shows the results of RT-PCR expression analysis of fad2mRM in white adipose tissue of transgenic pigs (B3, B12 and B19).
- M indicates the size marker.
- B-23, B-10, and B-10 are wild-type plants, and B-12, B-19, B-3 is a transgenic plant.
- Arrows indicate fad2-specific transcripts (378 bps).
- 5 yo is, two dogs of the transformer diethyl Nick blanking evening three animals (B- 12 and B- 19) expressed in m RNA levels of transgene in white adipose organization of was observed.
- FIG. 6 shows the results of Northern blot analysis of fad2 mRNA in the gene transfection of the B-12 strain.
- Poly A RNA (2 ng) was electrophoresed on a 1% agarose-formaldehyde gel and blotted on a nylon membrane. Arrows indicate fad2-specific transcripts (1.4 kbp).
- rRNA indicates a negative control
- WA from a TG mouse indicates a white adipose tissue (positive control) of the transgenic mouse whose expression was confirmed in Example 1
- Liver indicates a liver
- WA indicates a white adipose tissue.
- FIG. 6 it was confirmed that the transgene was transcribed specifically for white adipocytes. This indicates that the introduced fad2 gene is transcribed specifically in pig adipose tissue.
- Offspring were produced from two pigs (transgenic pigs,-1, early _1) having a ⁇ 12 fatty acid desaturase gene derived from two plants whose expression was confirmed. This confirms whether the introduced gene is stably transmitted to offspring. At the same time, it is convenient for pigs to which the gene has been transferred to examine whether the gene is expressed and the newly synthesized protein functions as an enzyme.
- the transgenic female transgenic plants into which the gene had been introduced were crossed with one wild-type male puta. Twelve offspring (survival-3 stillbirths-9) were obtained from the female transgenic buffalo, but all died due to breastfeeding in the mother.
- Fig. 7 shows the fatty acid composition ratio in the total lipids of the white adipose tissue of aP2 / fad2-introduced pigs (B-12 line) reared for 5 months on a normal diet and 8 weeks on an oleic acid diet.
- the vertical bar indicates the average value, and the error bar indicates the standard error.
- Statistical analysis was performed by Fisher's PLSD test after analysis of variance. From Fig. 7, the linoleic acid (18: 2 ⁇ -6) ratio (Mol%) of the total fatty acids in the transgenic B-12 strain was higher than that of the wild type at the start of administration of the oleic acid diet and at week 8. , Respectively, increased by 25 and 19% (paku 0.05). In addition, the oleic acid ratio was increased by 5 and 10%, respectively, in wild-type bushus 0.05).
- the linoleic acid composition ratio of 19-19 at the start of administration of the oleic acid diet and of white adipose tissue of wild-type female females was examined. At 12.9 and 10.3%, respectively, ⁇ -19 was It was expensive (paku 0.05).
- the increase in the linoleic acid ratio before the start of administration of the oleic acid diet was due to the fact that the lipids in white adipose tissue of the normal diet after administration had a very high content of oleic acid originally and were expressed in transgenic white adipose tissue. This is probably because FAD2 converted these oleic acids to linoleic acid.
- the cells are subcultured until a sufficient number of cells are obtained, and when induced with insulin or the like, the cells are differentiated again into fat cells and lipid droplets are accumulated in the cells (Japanese Patent Application Laid-Open No. 2000-2000). No. 836656). Since the gene used in pigs is linked to the aP2 promoter, it is not expressed in dedifferentiated precursor fat cells, but iad2 is expressed after differentiation induction. To this end, we considered that the function of the introduced fad2 could be examined in more detail by examining the fatty acid composition in the accumulated fat of the transfection-derived preadipocytes that had been induced to differentiate in vitro.
- dorsal fat cells from transgenic pigs and non-transfected pigs were collected using a biopsy needle.
- the cells were dispersed in the adipose tissue using a collagenase solution. Undigested tissue was removed by a filtration mesh to obtain isolated monocytic mature adipocytes.
- Transfer the cell suspension to a culture flask fill with 25 mM HEPES buf fered Dulbecco's Eagle minimum essential medium (FCS-DMEM) medium supplemented with 20% fetal calf serum, and invert the culture vessel upside down. Placed inside. In this way, the monocytic adipocytes float in the upper layer of the culture solution and adhere to the upper surface of the flask.
- FCS-DMEM Dulbecco's Eagle minimum essential medium
- Table 7 summarizes the results.
- the analysis of the fatty acid composition was performed three times using different cell samples from each cell group.
- a, b, c, and d in Table 7 a significant difference is observed between different signs in the same row.
- transgenic animal characterized in that the content of unsaturated fatty acids useful for health is increased by introducing a gene for a fatty acid desaturase into the animal and performing transformation. Methods were provided to increase the content of unsaturated fatty acids.
- the transgenic animal and the method of producing the transgenic animal of the present invention are particularly useful for the purpose of producing meat that is useful for health.
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JP2002357740A JP2003245070A (ja) | 2001-12-20 | 2002-12-10 | 脂肪酸不飽和化酵素を有する形質転換動物およびその作製方法 |
US10/543,886 US20060282907A1 (en) | 2003-01-30 | 2003-01-30 | Transgenic animal having fatty acid desaturase and method of producing the same |
PCT/JP2003/000930 WO2004066724A1 (ja) | 2001-12-20 | 2003-01-30 | 脂肪酸不飽和化酵素を有する形質転換動物およびその作製方法 |
EP03703094A EP1595447A4 (en) | 2003-01-30 | 2003-01-30 | Transgenic animal with fatty acid desaturase and method of preparation thereof |
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PCT/JP2003/000930 WO2004066724A1 (ja) | 2001-12-20 | 2003-01-30 | 脂肪酸不飽和化酵素を有する形質転換動物およびその作製方法 |
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US6686185B1 (en) * | 2000-03-07 | 2004-02-03 | Millennium Pharmaceuticals, Inc. | 25934, a novel fatty acid desaturase and uses therefor |
US7244874B2 (en) * | 2002-09-17 | 2007-07-17 | The Regents Of The University Of California | Stearoyl CoA desaturase transgenic non-human animals |
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Non-Patent Citations (4)
Title |
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DATABASE GENBANK 24 October 2002 (2002-10-24), TASAKA Y. ET AL.: "Spinacia oleracea FAD2 mRNA for delta-12 desaturase", XP002904425, Database accession no. (AB094415) * |
KOMANO TORU ET AL.: "Shoku shigen kenkyu no tenbo, shoku shigen dobutsu no riyo keishitsu no idenshi kogakuteki seigyo-idenshi kogaku ga design suru mirai no shoku shingen dobutsu", JAPANESE SCIENTIFIC MONTHLY, vol. 54, no. 5, 2001, pages 447 - 450, XP002904423 * |
MATSUMOTO K. ET AL.: "Production and analysis of transgenic mice bearing the higher plant gene", J. REPRODUC. DEV., vol. 46, no. SUPP., 2000, pages J59 - J63, XP002904424 * |
See also references of EP1595447A4 * |
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