JP6172699B2 - Hyperlipidemia model pig - Google Patents

Description

  The present invention relates to a low-density lipoprotein receptor (LDLR) gene knockout pig, its production method, and its use as a hyperlipidemia model.

  Hyperlipidemia (hypercholesterolemia) is one of lifestyle-related diseases, and it is known that LDLR plays an important role in regulating blood total cholesterol levels. Even in Japanese familial (hereditary) hypercholesterolemia (FH) patients, the relationship between blood cholesterol levels and at least four mutations in the LDLR gene is known (Non-patent Document 1). . Hyperlipidemia causes arteriosclerosis with prolonged symptoms. Atherosclerosis is a cause of heart disease and cerebrovascular disease among the three major causes of death in Japan. Therefore, hyperlipidemia and arteriosclerosis resulting therefrom are one of the important diseases for which development of treatment and prevention methods is required, and model animals for hyperlipidemia and arteriosclerosis are treated and prevented. Very useful in law research and development.

  However, model animals for hyperlipidemia and arteriosclerosis that sufficiently reproduce the pathology of human hyperlipidemia have not been obtained. Mice widely used as experimental animals have strong resistance to the onset of arteriosclerosis. For example, LDLR gene knockout mice showed a markedly high blood total cholesterol level under conditions given a high cholesterol diet. Under the conditions given, there was no significant increase in blood total cholesterol (Non-Patent Document 2). Moreover, although the mouse which knocked out the apolipoprotein E (ApoE) gene in addition to the LDLR gene showed a higher blood total cholesterol level than the LDLR gene knockout mouse, the blood total cholesterol level was similar to that of the ApoE gene knockout mouse. It was only shown (Non-Patent Document 3). Mice deficient in both the neuromedin U (NMU) gene and the ApoE gene have high blood low density lipoprotein (LDL) and very low density lipoprotein (VLDL) under conditions of normal feeding. ) Although it shows cholesterol level (Patent Document 1), a mouse knocked out with NMU, which is a neuropeptide, also exhibits symptoms such as overeating, obesity, hyperinsulinemia, hyperglycemia, etc., so a double knockout mouse with NMU gene and ApoE gene Hyperlipidemia is considered to be only one of the complex symptoms, and the relationship with LDLR is unknown. In the first place, the composition of blood LDL cholesterol and high density lipoprotein (HDL: High Density Lipoprotein) cholesterol in mice is different from that of human blood LDL cholesterol and HDL cholesterol. There is also a problem that it is not suitable for model animals. Therefore, there is currently no model mouse that can reproduce the pathology of human hyperlipidemia or arteriosclerosis.

  In addition, the WHHL rabbit (Watanabe Rabbit) and its related strain rabbits obtained as a result of breeding and selecting hyperlipidemic rabbits are used for research and development as hyperlipidemic model animals showing the pathological condition closest to human FH. I came. Later, it was revealed that WHHL rabbits had a genotype of LDLR hypofunction mutations, but since they were obtained by breeding selection, elements other than LDLR gene mutations may contribute strongly to the phenotype of WHHL rabbits. . WHHL rabbits have a marked increase in blood LDL cholesterol levels compared to control rabbits, and blood HDL cholesterol levels significantly decrease accordingly (Non-Patent Documents 4 and 5). In contrast, in human FH patients showing elevated LDL cholesterol levels, there is no significant decrease in HDL as seen in WHHL rabbits. Therefore, hyperlipidemic model rabbits including WHHL rabbits are not sufficient for reproduction of human FH pathology. Furthermore, since rabbits are herbivores in addition to being short-lived, they are not suitable for human hyperlipidemia model animals closely related to dietary habits. For example, in 1913, Russian pathologist Nikolai Anitkova announced that eggs were given to rabbits, which caused hyperlipidemia and arteriosclerosis. However, this is a symptom that was caused by feeding herbivorous rabbits in an experiment and eating eggs that were not normally eaten. It has become clear that. As can be seen from the above, rabbits that are herbivorous have a problem that they are not suitable for model animals of diseases closely related to human diet and lifestyle, hyperlipidemia and arteriosclerosis.

  As a recessive genotype (FH-r) that causes a phenotype of elevated blood cholesterol in pigs, the vicinity of the centromere of the second chromosome has been suggested, and a single-base missense mutation in the ligand-binding region of the LDLR gene has been identified. The direct relationship between such mutant genotypes and phenotypes is not clear (Non-Patent Document 6). Establishment of mouse embryonic stem cells (ES cells: Embryonic Stem cells) has enabled gene-targeted recombination (gene targeting). Various gene function analyzes have been advanced, and various human disease model mice have been produced by applying the technology. However, a test using a small animal such as a mouse having a short lifespan has a problem that it is difficult to use it for a long-term treatment experiment, a surgical treatment test, or the like. Therefore, the inventors conceived of creating a disease model animal using a pig having a long life and a large body, overcoming the problem that porcine ES cells were not established, Cell cloning pig production technology has been developed (Patent Documents 2 and 3), and by applying such technology, gene targeting can be performed even in pigs (Patent Documents 4 and 5). Patent Document 6 only describes the idea of using a pig having a mutation in the ApoE gene and / or LDLR gene as a model for the study of atherosclerosis, and Patent Document 7 also describes a part of the LDLR gene. Only a method for producing porcine cells in which is substituted with a drug resistance gene is described. Non-Patent Document 7 describes that mini-pigs were disrupted by gene targeting of the LDLR gene and observed that symptoms of hypercholesterolemia were observed, but the details are not clear. Mini pigs are smaller in size and have a smaller average number of offspring than ordinary meat pigs such as land races, so it takes a lot of time and labor to produce strains, and the number and organization of model pigs obtained. There is a problem in that the amount is limited.

Japanese Patent No. 4734523 Japanese Patent No. 4036356 Japanese Patent No. 4036608 JP 2008-220222 A JP 2010-110254 A WO2008 / 106982 publication WO2011 / 100505 gazette

Bujyo H. et al., J. Atheros. Thromb., 11, 146-151 (2004) Ishibashi S. et al., J. Clin. Invest., 92, 883-893 (1993) Ishibashi S. et al., Proc. Natl. Acad. Sci. USA, 91, 4431-4435 (1994) Tanzawa K. et al., FEBS Letter, 118, 81-84 (1980) Rosenfeld ME et al., Arteriosclerosis, 8, 338-347 (1988) Hasler-Rapacz J. et al., American Journal of Medical Genetics Volume 76, Issue 5, 379-386 (1998) Davis B. et al., Swine in Biomedical Research Conference 2011, S2-9 (2011)

  An object of the present invention is to provide a hyperlipidemia model animal that can sufficiently reproduce the pathology of human hyperlipidemia, and to treat and prevent hyperlipidemia using such a hyperlipidemia model animal. It is to provide a screening method for drugs.

  The inventors focused on pigs as omnivorous animals with large bodies and long lifespans, prepared LDLR gene knockout clone pigs, and examined the composition of plasma cholesterol fraction and neutral fat fraction. Since LDLR gene knockout mice do not show hyperlipidemia on a normal diet, contrary to the prediction that a single diet of LDLR gene will not show hyperlipidemia on a normal diet, But it showed symptoms of hyperlipidemia. Moreover, since the familial hyperlipidemia of humans could be sufficiently reproduced in the composition of plasma cholesterol fractions (VLDL, LDL, HDL), the LDLR gene knockout pig of the present invention is a conventional rodent or the like. As a result, the present invention has been completed with the confirmation that it is a superior hyperlipidemia model animal. The present invention is the result of joint research between Primetec Corporation and the National Institute for Agrobiological Sciences.

  That is, the present invention is characterized in that [1] the concentration of HDL cholesterol from which the function of all or part of the LDLR gene is deleted is similar to that of the control, and hypercholesterolemia is exhibited even in a normal diet. The hyperlipidemia model pig or [2] hyperlipidemia model pig according to [1] above, wherein the concentration of LDL cholesterol and VLDL cholesterol is higher than that of the control [3] The hyperlipidemia model pig according to [1] or [2], wherein the hypercholesterolemia is type IIb hereditary hyperlipidemia.

  The present invention can also be obtained by [4] enucleating a collected pig ovum and injecting into the enucleated ovum a pig somatic cell nucleus in which a part or all of the LDLR gene has been modified or deleted. A hyperlipidemia model pig according to any one of the above [1] to [3], or [5] a part or all of the LDLR gene, which is a somatic cell nuclear transfer cloned animal or its offspring The hyperlipidemic model pig according to [4] above, wherein the porcine somatic cell nucleus modified or deleted is a porcine somatic cell nucleus in which a part of the LDLR gene exon 4 is replaced with a neomycin resistance gene Or [6] a hyperlipidemic model pig according to [4] or [5] above, wherein the offspring of a somatic cell nuclear transfer cloned animal is produced by combining in vitro fertilization and artificial insemination techniques [7] Said [1 ~ About swine organ or cells derived from hyperlipidemia model pig according to any one of [6].

  [8] (a) a step of administering a test substance to the LDLR gene knockout pig or the cell derived from the LDLR gene knockout pig according to any one of [1] to [6]; b) a step of examining the concentration of LDL cholesterol or VLDL cholesterol in the test subject in step (a) or the presence or absence of arteriosclerosis; (c) the concentration of LDL cholesterol or VLDL cholesterol does not increase in step (b), or an artery Characterized by comprising the steps (a) to (c) of selecting a test substance as a candidate drug for the prevention or treatment of hyperlipidemia, using as an index that no sclerosis develops, A method for screening a prophylactic or therapeutic agent for lipemia, or [9] LDLR gene knockout pigs that lack all or part of the LDLR gene function, The concentration of DL cholesterol is comparable to the control, to a method also used as hyperlipidemia animal models showing hypercholesterolemia in a normal diet.

  ADVANTAGE OF THE INVENTION According to this invention, an LDLR gene knockout pig and its offspring can be provided, and it can be used as a hyperlipidemia model pig. In addition, a screening method for a therapeutic or preventive drug for hyperlipidemia using such an LDLR gene knockout pig can be provided.

The design of the PCR reaction and Southern blot for detection of LDLRex4TV vector construction and targeted recombination is shown. The wild-type LDLR locus used for the 5 ′ and 3 ′ homologous regions of the targeting vector (upper) and the target locus (lower) after homologous recombination are shown (exon; solid box, intron and adjacent region) ; Bold line). In the LDLRex4TV vector, the 5'-side homologous region was prepared with about 1 kb of the 5'-side exon 4 adjacent region, and the 3'-side homologous region was about 9 kb covering exons 4 to 9 (middle stage). Primer positions used for target recombination-specific PCR amplification are indicated by short arrows. The position of the probe used for the Southern blot is indicated by a short bar, and the expected size of the Southern blot band by HindIII or SphI digestion is shown for both wild type and target loci. (A, B) A photograph of a fetus obtained as a result of nuclear transfer and embryo transfer using LDLR KO cells, and (C) a new LDLR KO re-cloned pig. The blood cholesterol fractionation pattern (broken line) of LDLR KO cloned pigs is shown. 4 shows the results of 4-fraction analysis of blood cholesterol and neutral fat of LDLR KO porcine F2 generation. There is a significant difference between the different signs of ac (P <0.05). The results of 20-fraction analysis of blood cholesterol (A, B) and neutral fat (C, D) of LDLR KO porcine F2 generation are shown. There is a significant difference between the different signs of ac (P <0.05).

  The hyperlipidemic model pig of the present invention is a transgenic pig lacking the function of all or part of the LDLR gene, and has a HDL cholesterol concentration similar to that of the control, and is high even in a normal diet. Any LDLR gene knockout pig or its offspring showing cholesterolemia may be used, and the sex, age, and breed of the pig are not particularly limited. In the present specification, the term “hyperlipidemia model pig” or “LDLR gene knockout pig” also includes these progeny progeny. In the present invention, the control refers to a measured value in a pig that does not develop hyperlipidemia, such as a wild genotype pig fed a normal diet, that is, a normal diet having a general fat content. The normal food (ordinary feed) can be purchased as a commercial product, and the general fat content in this normal feed can include 2 to 5% fat content. The porcine LDLR gene is registered as Gene ID: 396801 in a database such as NCBI (National Center for Biotechnology Information), and a DNA sequence can be obtained from GenBank as accession number NC_010444.3, version GI: 347618792. Information such as mRNA sequence and amino acid sequence can also be obtained. Based on these sequences, DNA and amino acid properties and protein domain structure similarity can be used as indicators to obtain the DNA and amino acid sequences of the LDLR gene as appropriate using different databases. In addition, various information such as exon structure and SNP (single nucleotide polymorphism) can be obtained. In the present invention, examples of the LDLR gene include the gene ID: 396801, and the domain structure of proteins and genes recognized as LDLR genes from prior literature.

  In the present invention, the method for deleting all or part of the function of the LDLR gene is not particularly limited, and a control sequence such as a promoter of the porcine LDLR gene on the genome or a part or all of the LDLR gene is deleted or separated. The method of reversibly or irreversibly suppressing the function of all or part of the LDLR gene can be mentioned by substituting with the DNA sequence, thereby suppressing or inhibiting the expression of the functional LDLR gene. In addition, effects such as expression of a dominant negative mutant LDLR protein having a function lower than that of the wild type or a wild type can be obtained, and all or a part of the functions of the LDLR gene can be deleted. . A plurality of these may be used in combination, and a method of replacing a part of the LDLR gene with a drug resistance gene can be suitably exemplified. Such gene mutations may be homozygous or heterozygous. Good.

  Deletion of the function of all or part of the LDLR gene can be evaluated by examining LDLR protein expression, LDLR protein localization in cells and tissues, LDL endocytosis, and the like. The expression of LDLR protein can be examined by conventional methods such as quantitative PCR method and real-time PCR method for the expression of LDLR gene mRNA, and it is preferable to examine the amount of mRNA in multiple regions of LDLR gene mRNA. Primers and probes used in such methods can be appropriately designed and prepared based on the DNA sequence of the LDLR gene, or commercially available products can be obtained. Moreover, the expression of LDLR protein can also be detected using an anti-LDLR antibody, and can be examined by conventional molecular biological methods such as Western blotting and ELISA. The anti-LDLR antibody only needs to be able to recognize porcine LDLR protein, and a peptide can be designed based on the amino acid sequence of the porcine LDLR gene to produce an antibody, or a commercially available product can be obtained. For the localization of LDLR protein, observation samples such as fluorescent immunostaining and immunohistochemical staining using anti-LDLR antibody are prepared using cells and tissues, and the localization of LDLR protein in cells and tissues is determined using a confocal fluorescence microscope. It can be examined by a conventional molecular biology method such as observation by the like. Endocytosis of LDL is achieved by adding fluorescently labeled LDL while keeping cells and tissues at 4 ° C., binding fluorescently labeled LDL to LDLR on the cell surface, and then bringing the cells and tissues to 37 ° C. Endocytosis is initiated, and the uptake of LDLR or fluorescently labeled LDL into cells can be examined by conventional molecular biological methods such as observation with a fluorescence microscope. In addition, the behavior of the LDLR or LDL in real time can be observed using a confocal fluorescence microscope having a time lapse function.

  The gene mutation that deletes all or part of the functions of the LDLR gene may be performed by injecting a somatic cell nucleus having a genetic mutation into genomic DNA into an enucleated unfertilized egg to produce a somatic cell cloned pig. A transgenic pig can also be produced by causing a genetic mutation in the nuclear genomic DNA of a fertilized egg, and a method for producing a somatic cell cloned pig can be preferably exemplified. Progenies can be obtained using these somatic cell cloned pigs and transgenic pigs, and in this case, means such as in vitro fertilization and artificial insemination can be used in addition to normal mating. From the genomic DNA in which homologous recombination has occurred, the wild type protein of the target gene is not expressed, and it is considered that the function of the target gene is impaired.

  The control sequence such as the promoter of the porcine LDLR gene on the genome or the modification of the LDLR gene can be performed by using gene targeting method, zinc finger nuclease method, etc., and confirmation thereof is Southern blot analysis, PCR reaction, restriction enzyme reaction. It can be carried out using conventional genetic engineering methods such as treatment with mismatch-specific nuclease and electrophoresis. In the gene targeting method, a targeting vector DNA is introduced into a cell, and a part of the target gene is replaced with a sequence such as a resistance gene by homologous recombination between the genomic DNA and the targeting vector DNA, thereby replacing the normal target gene sequence. This is a method for detecting homologous recombinants by destroying and using the function of such resistance genes as an index. The targeting vector consists of a DNA sequence at the 5 ′ end of the target site in the target gene on the genome (5 ′ homologous region), a drug resistance gene expression cassette, and a DNA at the 3 ′ end of the target site in the target gene on the genome. What is necessary is just to have a sequence (3′-side homologous region) in order, and in addition, a sequence necessary for functioning as a vector may be provided. The target site is not particularly limited, and is preferably an exon, and particularly preferably exon 4. The length of the 5 ′ homologous region and the 3 ′ homologous region is not particularly limited as long as homologous recombination occurs, and is preferably 100 bases (b; base) to 20 kilobases (kb), more preferably 200 b to 15 kb, most preferably Preferably it is 500b-10kb. The targeting vector may be introduced into the cell as DNA linearized by restriction enzyme treatment, etc., and the introduction method into the cell uses the calcium phosphate method, lipofection method, electroporation method or commercially available transfection reagent as appropriate. can do.

  In the zinc finger nuclease method, the genome sequence can be easily edited by causing a double strand break (DSB) to occur in the target gene of the genomic DNA using a chimeric zinc finger nuclease. Cells / organisms in which deletion, insertion, or modification (substitution) of the target gene has occurred can be obtained (see JP-T-2005-519631, JP-T 2006-518372, JP-T 2009-538141, etc.). That is, when DSB is induced, a mutation is introduced in the subsequent DNA repair process, or homologous recombination is induced with high efficiency, and a part of the sequence of the target gene is replaced with a sequence such as a resistance gene, Normal protein expression is impaired. Here, the chimeric zinc finger nuclease is a chimeric protein in which a zinc finger protein having DNA binding ability and a nuclease domain of type II restriction enzyme FokI are fused, and the zinc finger protein is appropriately selected according to the DSB target site of the target gene. Can be designed. Nuclease FokI forms heterodimers and cleaves DNA. In the vicinity of the DSB target site of genomic DNA, a zinc finger target sequence is set in each complementary strand, and a chimeric zinc finger nuclease that functions as a pair is placed in a cell. To express within. Such a chimeric zinc finger nuclease can be introduced into cells as mRNA or an expression vector, and calcium phosphate method, lipofection method, electroporation method, commercially available transfection reagent, etc. can be appropriately used for introduction into cells. The chimeric zinc finger nuclease can be produced using a gene recombination technique, or a commercial product such as CompoZr (registered trademark) Zinc Finger Nuclease (manufactured by Sigma-Aldrich) can be obtained.

  Furthermore, the LDLR gene knockout may occur from the fertilized egg or the early stage of development, or may occur during the development or after growth by being induced in a specific time or site using the Cre-loxP system or the like. Using a vector, a somatic cell clone is created using a somatic cell nucleus in which the LDLR gene function has been deleted by replacing exon 4 of the LDLR gene on the genome with a neomycin resistance gene by homologous recombination. It can be illustrated. As a targeting vector for performing the homologous recombination, a 5 ′ homologous region including the 5 ′ region of exon 4 of the genomic LDLR base sequence is present on the 5 ′ side of the neomycin resistance gene expression cassette, and exon 4 is present on the 3 ′ side. The 3 ′ side homologous region including the 3 ′ side region is preferable. The position and length of these homologous regions can be appropriately adjusted, and the genomic LDLR base is located on the 5 ′ side of the neomycin resistance gene expression cassette. An example in which an approximately 1 kb 5 ′ homologous region extending from exon 3 to exon 4 of the sequence and an approximately 9 kb 3 ′ homologous region extending from exon 4 to 9 on the 3 ′ side is preferable.

  The hyperlipidemia model pig of the present invention may be of any genotype having a heterozygous or homologous LDLR gene knockout chromosome, and is preferably a homologous LDLR gene knockout genotype. The LDLR gene knockout pig is enucleated from the collected pig egg and injected with a somatic cell nucleus having a genomic DNA in which a part or all of the LDLR gene is modified or deleted. By doing so, it can be produced as a somatic cell nuclear transfer cloned animal. If the somatic cell nuclear transfer cloned pig has a normal breeding ability, it can be crossed with a wild type or the above somatic nuclear transfer cloned pig, etc. to obtain progeny, and similarly, the strain can be maintained by the normal breeding method. Progeny offspring can be obtained. In addition, when the health status of the somatic cell nuclear transfer cloned pig is unfavorable and not suitable for breeding by a normal method, in vitro fertilization and artificial insemination using a gamete, preferably a sperm, collected from the somatic cell nuclear transplant cloned pig The above-mentioned LDLR gene knockout pig can also be produced by combining the above methods to obtain a progeny of a cloned pig. The LDLR gene knockout pig and the hyperlipemia-producing pig strain of the present invention can be maintained by the in vitro fertilization and artificial insemination methods, or the strain can be maintained by a normal breeding method to obtain progeny offspring. As long as both are transgenic pigs that lack all or part of the function of the LDLR gene, their progeny are also included in the LDLR gene knockout pig and hyperlipidemia model pig of the present invention. .

  As hypercholesterolemia in the present invention, the blood total cholesterol concentration should be higher than that of the control, and the blood total cholesterol concentration of the hyperlipidemic pig is 1.5 times or more, preferably 3 times or more of the control, More preferred examples are four times or more. Further, in addition to the blood cholesterol concentration, the blood total neutral fat concentration of the hyperlipidemic pig is preferably higher than that of the control. In the present invention, the meanings of hypercholesterolemia and hyperlipidemia are essentially the same. Human hyperlipidemia can be classified into types I, IIa, IIb, III, IV, and V based on the amount of lipid components in the blood. Type IIb has higher levels of VLDH cholesterol and LDL cholesterol than healthy subjects. The patient has hyperlipidemia with a cholesterol level similar to that of a healthy person. The hyperlipidemia model pig of the present invention has a blood fat component composition similar to that of IIb hereditary hyperlipidemia, in which the VLDH cholesterol concentration and the LDL cholesterol concentration are higher than those of the control, and the HDL cholesterol concentration is similar to that of the control. Since it shows, it can also be set as the model pig of type IIb hereditary hyperlipidemia.

  In the present invention, the concentration of HDL cholesterol, LDL cholesterol, or VLDL cholesterol can be measured as the HDL, LDL, or VLDL concentration in the cholesterol category of blood-derived samples such as plasma and serum. Similarly, HDL triglyceride, LDL triglyceride, Or as a density | concentration of VLDL triglyceride, it can measure as a HDL, LDL, and VLDL density | concentration in the triglyceride (neutral fat) division of blood-derived samples, such as plasma and serum. Blood lipids can be measured by a conventional method as appropriate. Data can be obtained by providing a sample to a measuring agent, or can be measured using a measuring instrument. For the measurement of lipids in blood, whole blood may be used, or a serum sample or plasma sample, or a fractionated sample may be used as appropriate. These are anticoagulants such as heparin and preservatives unless they interfere with the measurement. An antifungal agent, a pH adjuster, a buffer solution, and the like can be appropriately added. For example, when each component of VLDL, LDL, and HDL is quantified, a serum sample is first fractionated into each component, and for each fraction, Determiner L TCII and Determiner TC-555 (manufactured by Kyowa Medex Co., Ltd.), Quantification by an enzymatic method can be performed using an enzymatic method kit such as Aqua Autokinos T-CHO Reagent (manufactured by Kainos Co., Ltd.). Moreover, in order to fractionate each component of VLDL, LDL, and HDL, a gel filtration column capable of analyzing lipoproteins in serum, such as TSKgel LipopropakXL, TSKgel Lipopropak (manufactured by Tosoh Corporation), etc., is used. It can also be analyzed by chromatography (HPLC) methods.

  In the present invention, the concentration of HDL cholesterol in the blood of the hyperlipidemic model pig of the present invention may be the same as that in the control, and the HDL concentration in the plasma cholesterol category of the LDLR gene knockout pig is 0.7 to It is 1.5 times, preferably 0.8 to 1.3 times, more preferably 0.9 to 1.2 times. Further, the LDL cholesterol and VLDL cholesterol concentrations in the hyperlipidemic model pig of the present invention should be higher than those in the control, and the concentrations of LDL and VLDL in the plasma cholesterol category are 1.5 times or more, preferably 2 It is preferable to exemplify that it is twice or more, more preferably 3 times or more, and further preferably 4 times or more. Although there are no particular limitations on the concentration of blood lipid components such as total cholesterol, total neutral fat, HDL cholesterol, LDL cholesterol, and VLDL cholesterol in the blood of the hyperlipidemic model pig of the present invention, and blood composition, An example in which the blood LDL triglyceride of the hyperlipidemia model pig of the invention is higher than that of the control can be preferably exemplified. In addition, these blood cholesterol levels vary depending on age, breeding environment, and type of food, and symptoms of hyperlipidemia progress due to feeding such as aging and high cholesterol diet and exercise restriction. Therefore, even if the hyperlipidemia model pig of the present invention does not show hyperlipidemia immediately after birth or during the growth process, it may be hypercholesterolemia at the time of measuring cholesterol in blood, preferably blood at the time of measurement. The concentration of HDL cholesterol in the middle is the same as that of the control, more preferably, the concentration of HDL cholesterol in the blood is the same as that of the control at the time of measurement, and hypercholesterolemia is shown in the measurement of LDL cholesterol and VLDL cholesterol. An example can be given.

  In the present invention, the normal pig food may be a normal feed generally given when pigs are raised, and commercially available products can be purchased. For example, feed names “Etsu” and “Hatsutsutsu” ( JA Zen-no-Mori), feed names "Friend U" and "Mother Breed" (made by Shimizu Port Feed Co., Ltd.), etc., with a fat content of 0.01-8%, more preferably 0.1-7% More preferably, 1 to 5% of feed can be mentioned. On the other hand, the high-cholesterol diet is not particularly limited as long as it has a fat content higher than that of a normal diet, and can be prepared by mixing lard, high-fat edible oil, fat or the like as cholesterol in the regular diet. The cholesterol content of the high cholesterol diet is preferably 0.5 to 5%, more preferably 2%, and the fat content is 13 to 50%, more preferably 14 to 30%, still more preferably 15%. -25% can be mentioned. Pigs fed a normal diet have a total cholesterol level in the blood of 50-100 mg / dl, whereas pigs fed a high cholesterol diet have a high fat content in which the total cholesterol level in the blood is 500-800 mg / dl. (See Dixon JL. Et al., Arterioscler Thromb Vasc Biol. 1999, 19: 2981-2992; M. Michael Swindle, Swine in the Laboratory second edition, 314-315, CRC Press. ).

  As one embodiment of the present invention, an LDLR gene knockout pig lacking the function of all or part of the LDLR gene has a high HDL cholesterol concentration comparable to that of a control and exhibits high cholesterol even in a normal diet. It can be set as the method of using as a lipemia model animal. An LDLR gene knockout pig lacking all or part of the functions of the LDLR gene of the present invention has a HDL cholesterol concentration similar to that of a control, and a hyperlipidemia model showing hypercholesterolemia even in a normal diet The method used as an animal is not particularly limited, and is used for the determination of hyperlipidemia and diseases caused by hyperlipidemia, such as arteriosclerosis, myocardial infarction, cerebral infarction, etc. The use as a practice base to perform can be preferably exemplified.

  The LDLR gene knockout pig and the hyperlipidemia model pig of the present invention are likely to develop arteriosclerosis and myocardial infarction even under conditions given a normal diet, and appropriately feed a high cholesterol diet to induce these symptoms. You can also. Also, pigs can be aged because such symptoms are likely to develop. The LDLR gene knockout pig or hyperlipidemia model pig of the present invention fed with a normal diet or a high cholesterol diet is used for the determination of hyperlipidemia, determination of the presence or absence of arteriosclerosis or myocardial infarction, arteriosclerosis or myocardial infarction. It can be used as a practice model pig for the development of surgical methods and surgical instruments and for the practice of surgical techniques. Although it does not restrict | limit especially as said surgery method, In the treatment of diseases, such as myocardial infarction and cerebral infarction resulting from hyperlipidemia, such as stent placement, bypass surgery, catheter surgery, carotid endarterectomy (CEA), etc. Examples of surgical instruments include stents and catheters. Practice of the determination of the presence or absence of hyperlipidemia, arteriosclerosis and myocardial infarction can be performed using the individual pigs for practice and their blood, blood vessels and tissues. For example, measurement results of heart rate, pulse rate, blood pressure, etc., and blood glucose level, red blood cell level, white blood cell level, blood LDL cholesterol level, blood HDL cholesterol level, blood VLDL cholesterol level, etc. Practice of determining the presence or absence of hyperlipidemia, arteriosclerosis and myocardial infarction can be performed from blood test results and test results such as ultrasonic examination (echo) and magnetic resonance imaging (MRI). In addition, body fluids such as blood, blood vessels, and tissues such as blood collected from such training model pigs are also subjected to blood tests, dissections, histochemical staining, etc., and hyperlipidemia, arteriosclerosis, myocardial infarction Can practice judgment. In addition, the LDLR gene knockout pig or hyperlipidemia model pig of the present invention that has developed arteriosclerosis or myocardial infarction can be used as an arteriosclerosis model pig or a myocardial infarction model pig. It can be used for practice in humans to make such a determination using a blood vessel, a tissue having such a blood vessel, or a heart that has undergone myocardial infarction. Similarly, it is possible to develop a method for determining hyperlipidemia and a disease caused by hyperlipidemia, a therapeutic method, and a surgical method.

  In addition, an LDLR gene knockout pig in which the function of all or part of the LDLR gene of the present invention has been deleted is a hyperlipidemia in which the concentration of HDL cholesterol is similar to that of the control and hypercholesterolemia is exhibited even in a normal diet. Examples of the method for use as a disease model animal include an example in which an LDLR gene knockout pig or a hyperlipidemia model pig lacking the function of all or part of the LDLR gene is used as a drug evaluation animal. A therapeutic drug candidate drug for a disease caused by hyperlipidemia or hyperlipidemia is administered to the LDLR gene knockout pig or hyperlipidemia model pig of the present invention fed with a normal diet or a high cholesterol diet, Using the improvement of cholesterol level, arteriosclerosis, myocardial infarction pathology, or the fact that they do not worsen as an index, the efficacy of the therapeutic drug candidate drug can be examined, and simultaneously its side effects can be investigated.

  Since the LDLR gene knockout pig of the present invention has high LDL cholesterol and VLDL cholesterol concentrations compared to the control, and the HDL cholesterol concentration is comparable to the control, and also shows hypercholesterolemia in a normal diet, It closely reproduces the pathology of human type IIb hereditary hyperlipidemia and is useful as a model animal of type IIb hereditary hyperlipidemia. The organ or cell derived from the LDLR gene knockout pig or hyperlipidemia model pig of the present invention may be any biological sample derived from the LDLR gene knockout pig or hyperlipidemia model pig of the present invention. In addition to living tissues in the head, neck, chest, abdomen, legs, such as organs, bones, body fluids, etc., cells can be mentioned, such cells include epithelial cells, hepatocytes, fibroblasts, kidney cells, etc. Examples include somatic cells, nerve cells, immune cells, ES cells, primordial germ cells, sperm and eggs, and primary cultured cells of these cells or cell lines derived from these cells, that is, cells grown from these cells. Including.

As a screening method for a prophylactic or therapeutic agent for hyperlipidemia of the present invention,
(A) a step of administering a test substance to the LDLR gene knockout pig or cells derived from the LDLR gene knockout pig;
(B) a step of examining the concentration of LDL cholesterol or VLDL cholesterol in the test subject in step (a), or the presence or absence of arteriosclerosis;
(C) In the step (b), the test substance is selected as a candidate drug for the prevention or treatment of hyperlipidemia using as an index that the concentration of LDL cholesterol or VLDL cholesterol does not increase or does not develop arteriosclerosis Process;
Steps (a) to (c) may be included, and other steps may be included. In addition, a control such as a wild-type pig can be used in combination as appropriate.

  The step (a) of the screening method of the present invention may be a step of administering a test substance to the LDLR gene knockout pig of the present invention or a cell derived from the LDLR gene knockout pig, which is the test subject. You can choose. Examples of test substances include chemical substances, proteins, peptides, steroids, etc. These can be prepared by synthesis, extraction, etc., or commercially available products can be purchased, and commercially available libraries can be used. it can. Moreover, the administration method of the test substance to the LDLR gene knockout pig of the present invention or the cells derived from the LDLR gene knockout pig can be appropriately selected. Examples of the LDLR gene knockout pig include oral, injection, infusion, and application methods. Examples of cells derived from LDLR gene knockout pigs include addition to cell culture medium and administration into cells using transfection reagents or electroporation. The administration period, the number of administrations, and the dosage of the test substance can also be appropriately selected depending on the size, age, sex, symptoms, etc. of the administration subject. The breeding environment and age of the test subject pig, the culture conditions of the test subject cells, and the like can be appropriately selected. Although a cholesterol diet may be fed as the feed for the pigs to be tested, the LDLR gene knockout pig of the present invention has the property of exhibiting hyperlipidemia in a normal diet, and therefore can be fed a normal diet .

  Step (b) of the screening method of the present invention is only required to be able to examine the degree of hyperlipidemia in the test subject pig of step (a), and the concentration of LDL or VLDL in the plasma cholesterol category or arteriosclerosis An example of examining the presence or absence of LDL, HDL, and VLDL in the total cholesterol concentration in plasma, the concentration of HDL in the cholesterol category, the total triglyceride concentration and the triglyceride category, and HDL In addition, other test items such as blood glucose level, red blood cell quantity, white blood cell quantity, etc. may be examined. In addition, arteriosclerosis can be examined histochemically by ultrasonography (echo), magnetic resonance imaging (MRI) or the like, or by collecting a vascular tissue sample or the like from a subject to be examined. When the test subject is a cell derived from an LDLR gene knockout pig, its growth rate and metabolic rate, intracellular LDL cholesterol and VLDL cholesterol amount, etc. are examined by a general biochemical and cell biological technique. be able to.

  Step (c) of the screening method of the present invention is based on the measurement result obtained in step (b), with the LDL cholesterol or VLDL cholesterol concentration not increasing or developing the arteriosclerosis as an index. Any process may be used as long as it is selected as a candidate drug for the prevention and / or treatment of lipemia, and the health status of the test subject, the results of other test items, and the like can also be referred to. In particular, when the concentration of LDL cholesterol or VLDL cholesterol decreases, it can be determined that the drug is promising as a candidate drug for the prevention and / or treatment of hyperlipidemia.

  As another aspect of the screening method of the present invention, tissues and cells derived from the LDLR gene knockout pig of the present invention can be appropriately used for screening according to the purpose. That is, it is difficult to obtain reliable screening and data in cells and tissues derived from a hyperlipidemia model WHHL rabbit that is selected by conventional line breeding and the causal relationship between phenotype and genotype is not clear, In addition, although rabbits have different problems from humans, such as lack of HDL-to-LDL converting enzyme, the LDLR gene knockout pig of the present invention has a mutation only in the LDLR gene, so screening that focuses on the LDLR gene, LDLR and cells It is useful in medical and biological research such as functional analysis. For example, the cells derived from the LDLR gene knockout pig of the present invention can be used for screening with the change of the signal factor to be analyzed as an index. Specifically, (a ′) the cells derived from the LDLR gene knockout pig of the present invention are used. A step of administering a test substance to a cell or tissue; (b ′) a step of examining a change in a signal factor in the test subject in step (a ′); (c ′) a change in the signal factor in step (b ′). Screening including steps (a ′) to (c ′) of selecting a test substance different from the control can be performed, and other steps may be included as appropriate. Factors involved in biological phenomena involving LDLR and lipoprotein metabolism and regulators of signal factors can be identified by screening.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

<Method: Production of LDLR KO cloned pig>
1) Structure of LDLR targeting vector (vector name: LDLRex4TV)
The LDLR genomic region (ACCESSION No. NC — 010444), 62049795 to 62063187 genomic region for the production of LDLRex4TV vector was isolated from the genomic DNA of a pig LWD (Landlace × Large Yorkshire × Duroc) ternary hybrid. As shown in FIG. 1, the LDLRex4TV vector comprises an approximately 1 Kb 5 ′ homologous region from exon 3 to exon 4 and an approximately 9 kb 3 ′ homologous region from exon 4 to 9 of the genomic LDLR base sequence. (Boer, PH et al. Polymorphisms in the coding and noncodingregions of murine Pgk-1 alleles. Biochemical genetics 28, 299-308 (1990)) and ligated to both sides of the PGK-neo-p (A) cassette, Furthermore, outside of the 5 'homologous region, pMC1-Dtp- (A) (Taniguchi, M. et al. Disruption of semaphorin III / D gene causes severe abnormality in peripheral nerve projection. Neuron 19, 519-530 (1997)) The cut out MC1-DTA-p (A) cassette is placed. The 5 ′ homologous region was PCR amplified using PCR primer names: LDLRex3F2: 5′-TCAGCAGGACTGCGAGAATG-3 ′ and LDLRex4R3: 5′-CATCTTGGGAGCACGTCTTG-3 ′. The 3 ′ homologous region was PCR amplified using the primer names LDLRex4F1: 5′-AAGTCTGACGAGGAGAACTG-3 ′ and LDLRR3: 5′-AGAAGAGGTAGGCGATGGTG-3 ′. This targeting vector (vector name: LDLRex4TV) was linearized by NotI restriction enzyme digestion before gene introduction by electroporation.

2) Collection of fetal fibroblasts After mating with Landrace (L) female x Landrace (L) male, fetuses (L) on the 68th day of pregnancy and fetuses (LW) on the 62nd day of pregnancy were collected. For the collected fetus, muscle tissue was collected with scissors and minced. The suspension was suspended in 10 ml of 0.25% trypsin + 0.04% EDTA-2Na solution per 1 g of minced fetal tissue and refrigerated overnight (4 ° C.). After removing the supernatant by centrifugation (1500 rpm, 5 minutes), it was treated in a water bath kept at 37 ° C. for 20-30 minutes. After the heat treatment, 10 ml of DMEM + 10% FCS per 1 g of fetal tissue was added and pipetted, followed by filtration with a Celes toilet. After centrifuging the filtrate, the supernatant was removed, and 10 ml of DMEM + 10% FCS was added to the precipitate and resuspended. The number of cells was counted, adjusted to an appropriate amount with DMEM + 10% FCS, and then seeded in a 75 cm ² cell culture flask. The seeded cells were cultured at 37 ° C. with 5% CO ₂ until they became confluent and used for gene transfer by the following electroporation.

3) Gene transfer and G418 drug selection (production of LDLR gene knockout cells)
The separated and proliferated fetal fibroblasts were detached with 0.25% trypsin-0.04% EDTA-2Na solution and suspended in DMEM + 10% FCS. After the cell suspension was centrifuged (1500 rpm, 5 minutes), the supernatant was removed and resuspended in cold HBS. The resuspension was centrifuged again to remove the supernatant. It adjusted with HBS so that the cell number might be set to 2.5 * 10 < ⁷ > cells / ml. The cell solution 0.4 ml (1.0 × 10 ⁷ cells / ml) and 100 μl of the prepared DNA solution (5 nM linearized targeting vector was added, FIG. 1) were added to the cuvette and mixed well. For gene transfer, an electroporation apparatus was used with pulses of 300 V, 950 μF, and resistance ∞. After applying the pulse, it was allowed to stand at room temperature for 10 minutes. The stationary cells were suspended in DMEM + 10% FCS, and then divided into three 10 cm dishes and cultured. Forty-eight hours after the pulse, 800 μg / ml neomycin (G418) was added to the culture solution to select a G418-resistant cell line. After drug selection for 7-12 days, G418-resistant cell colonies were separated with a filter paper disk and transferred to a 24-well cell culture dish. After the transferred cells reached confluence, they were detached by trypsin treatment. Half of the detached cells were used for PCR analysis, and the rest were left intact.

4) Detection of targeting by PCR analysis The cells were resuspended in 50 μl of ultrapure water containing 200 g / ml of proteinase K, incubated at 55 ° C. for 3 hours, and then heated at 95 ° C. for 10 minutes. Was deactivated. PCR analysis was performed using AmpliTaqGold (registered trademark) (Applied Biosystems) by collecting 1 μl as a template from a 50 μl sample digested in a 20 μl amount of the reaction solution. The PCR cycle parameters were as follows: 94 ° C for 10 minutes, 94 ° C for 30 seconds, 60 ° C for 25 seconds, 72 ° C for 1 minute 15 seconds in 35 cycles, and finally 72 ° C for 7 minutes. Processed. The primers used were FP2 (5′-ACCAAATTAAGGGCCAGCTC-3 ′) and LDLRex3F4 (5′-GCAAGATAGGGGACTTTAGC-3 ′). This primer set amplifies a 1.1 kb mutant allele specific product. PCR on the purified DNA was performed in the same manner except that 100 ng of purified DNA was used as a template.

5) Egg collection method by hormonal treatment Use of sows before spring activation 160 to 194 days of age of ternary hybrids (LWD) of Landrace (L) and Landrace, Large Yorkshire (W), Duroc (D) It was. The test pigs were administered 1500 IU of eCG (Pimex, Sankyo Pharmaceutical Co., Ltd.), and hCG (Puperogen, Sankyo Pharmaceutical Co., Ltd.) was administered 500 IU 72 hours later. At 48 hours after hCG administration, the mice were sacrificed, and connective tissues of the ovaries, fallopian tubes, and uterus were removed. In addition, eggs collected from sows that reached sexual maturity were subjected to artificial insemination using male-diluted semen at the time of estrus, and miscarriage treatment was performed 21-50 days after pregnancy. Five days before the planned nuclear transfer, about 0.2 mg of PGF2α analog cloprosterol (Planate, Takeda Pharmaceutical Co., Ltd .: 2 ml amount) was administered intramuscularly. 24 hours later, PGF2α analog cloprosterol was similarly injected, and at the same time, eCG (Pimex, Sankyo Pharmaceutical Co., Ltd.) was administered into the buttocks muscles at 1500 IU. 72 hours after administration of PGF2α + eCG, 500 IU of hCG (Puperogen, Sankyo Pharmaceutical Co., Ltd.) was administered. The mice were sacrificed 46 hours after the administration of hCG, and connective tissues of the ovaries, fallopian tubes and uterus were removed.

6) Preparation of mature oocytes in the body The removed connective tissue of the ovary, fallopian tube, and uterus was immediately brought back to the laboratory, and the perfusate (PBS (Ca ²⁺ , Mg ²⁺ free, manufactured by TAKARA) + 0.1% bovine serum albumin ( BSA, Sigma, Cat. No. A-4503) + 1 ml of added antibiotic per 100 ml (Antibiotic, Antimycotic, Sigma, Cat. No. A-7292))) . The obtained ovum was made naked with cumulus cells using a perfusion solution containing 0.1% hyaluronidase, and an egg with uniform egg cytoplasm was selected, and then washed in a perfusion solution not containing hyaluronidase. Thereafter, in vitro culture was performed using the PZM-3 solution until it was tested in an incubator (38.5 ° C., 5% CO ₂ , 5% O ₂ , 90% N ₂ ).

7) Ovulation method of ovarian follicular ovum from slaughterhouse ovaries were collected from gilts in the slaughterhouse before the start of spring slaughtered for meat use, and kept in the laboratory with PBS (-) solution at 35 ° C. I took it home. The ovaries brought home were immediately washed with PBS (−) kept at 37 ° C., and ovarian connective tissue was removed, and then egg collection was performed. Follicles with a diameter of 3-6 mm were selected and aspirated with a 10 ml syringe with a 16G needle. The aspirated follicular fluid was collected in a 50 ml disposable tube, allowed to stand at room temperature for 5 minutes or more, and then the supernatant was removed. Add suspension of TALP-Hepes to the precipitate containing the egg, confirm that the precipitate has accumulated at the bottom of the tube, and remove the supernatant with a Pasteur pipette so as not to aspirate the precipitate. did. This washing operation was repeated 2-3 times. Selection and egg collection of cumulus cell ovum complex (COC) in which cumulus cells are densely attached in three or more layers and wall side granule layer cell-cumulus cell ovum complex (GCOC) in which wall side granule layer cells are attached to COC Did.

8) In vitro maturation culture 100 μl of the modified NCSU37 was dispensed at about 15 to 20 cells / well, and matured using 6-well dishes covered with 100 μl liquid paraffin oil. Modified NCSU-37 is 10% porcine follicular fluid, 0.6 mM cysteine, 50 μM β-mercaptoethanol, 1 mM dibutyryl cAMP, 10 IU / ml PMSG (Pimex; 1000 IU, Sankyo Pharmaceutical Co., Ltd.), 10 IU / ml hCG (properogen; 1500 IU, manufactured by Sankyo Pharmaceutical Co., Ltd.). After culturing for 20 to 22 hours, it was transferred to a modified NCSU 37 without hormones and dibutyryl cAMP and cultured until the test. The mature culture was performed at 39 ° C., and the gas phase conditions were 5% CO ₂ , 5% O ₂ and 90% N ₂ . The mature culture time was 42-44 hours, and only mature eggs (first polar body released eggs) were used for the test.

9) Oocyte nucleus removal operation (enucleation operation)
Prior to the enucleation operation, the slaughtered unfertilized ovum was transferred to a PZM3 solution containing 5 μg / ml of cytochalasin B (Sigma: Cat. No. C-6762) and treated for 15 minutes or more. The enucleation operation was also carried out in a drop made with PZM3 solution containing cytochalasin B at the same concentration (prepared with 50 μl of a drop near the center of a 10 cm plastic petri dish and covered with 20 ml of mineral oil). The egg was held with a holding pipette so that the first polar body was positioned from 12:00 to 3 o'clock. The cytoplasm of the first polar body is ¼ to ３ by a pipette for enucleation (outer diameter 25-30 μm, tip is polished at an angle of 30 to 45 degrees) attached to a piezo micromanipulator (manufactured by Prime Tech). An amount was aspirated. When the operability of the enucleating pipette deteriorated, washing was performed by repeated suction and discharge with a 20% PVP solution (Sigma: Cat. No. PVP-360) drop. Immediately after the enucleation operation is completed, transfer to a PZM3 solution containing no cytochalasin B and perform washing. For washing, cytochalasin B was removed by carefully washing twice or more with a 35 mm dish (Falcon 1008) containing 3 ml of PZM3 solution. Thereafter, until the injection operation, it was transferred to a PZM 3 liquid drop (35 mm dish: a 100 μl drop was prepared on Falcon 3001 and covered with 4 ml mineral oil) and cultured in an incubator.

10) Preparation of Donor Recombinant Cells Male fetal fibroblasts that were determined to have the LDLR gene knocked out by PCR analysis were used as nuclear transfer donor cells. As the culture solution, DMEM + 10% FCS was used, and the cells were transplanted 0 to 5 times. In accordance with the scheduled day of nuclear transfer, after transplanting, the culture medium was not changed from the confluent state and left for 3 to 14 days to synchronize the cell cycle with the G1 / G0 phase. Donor somatic cells were prepared approximately 1 hour before being used for nuclear transfer. In the cell treatment, after removing the culture solution from the culture vessel, washing was performed 3 times or more with PBS (−), and cell detachment treatment was performed with 0.25% trypsin + 0.04% EDTA-2Na solution. 10% FCS-added DMEM was added to the detached cells and suspended, followed by centrifugation (1,500 rpm, 5 minutes, room temperature). After centrifugation, the supernatant was removed, an appropriate amount of PZM3 solution was added, suspended, and left at room temperature until used for nuclear transfer. At the time of somatic cell nucleus injection operation, an appropriate number of cells was added to the drop of the injection operation chamber for use.

11) Injection procedure of somatic cell nucleus Add an appropriate amount of donor cells to the drop of the injection chamber (10 cm petri dish with 50 μl of PZM3 solution and cover with 20 ml of mineral oil), and remove 50-100 enucleated eggs. I put it in and operated it. For the injection operation, a pipette for somatic cell nucleus injection (outer diameter 7-10 μm, blunt end) was used in a piezo micromanipulator. Before the injection pipette was attached to the piezo micromanipulator, it was used by filling Fluorinert with a width of 3-5 mm at a position 5 mm from the rear end of the pipette. When using the pipette attached to the piezo, the pipette was filled with culture solution, florinate, air, and mineral oil in that order. Before performing the injection operation, the injection pipette was always washed in the 20% PVP liquid drop (pipetting and the pipette tip into and out of the PVP drop). The floating somatic cells were aspirated and pipetted several times to break the cell membrane and injected into the enucleated oocyte cytoplasm held with a holding pipette. Oocytes injected with somatic cell nuclei were cultured in an incubator until the activation treatment time (1-3 hours).

12) Ovum Activation Treatment The activation treatment of the ovum injected with somatic cell nuclei was performed by direct current pulse stimulation (SSH-2, manufactured by Shimadzu Corporation) under the conditions of 1.5 kV / cm 100 μsec × 1 time. A stainless steel wire electrode having a width of 2 mm was used for the chamber, and a 0.28 M mannitol solution containing 0.05 mM CaCl ₂ , 0.1 mM MgSO ₄ and 0.02% BSA was used for the electrolyte solution for electrical stimulation.

13) Inhibition of the second polar body release of the activated ovum The activated ovum was cultured in PZM3 solution containing 5 μg / ml cytochalasin B (Sigma: Cat. No. C-6762) for 2 hours. Treatment to suppress body release was performed.

14) In Vitro Culture of Activated Eggs Eggs that had been diploid treated after activation were subjected to in vitro culture in PZM3 solution, and only split embryos were used for embryo transfer on the second day after the activation treatment.

15) Synchronization of temporary stomach by hormonal treatment and embryo transfer method Synchronization of temporary stomach was performed by artificial abortion treatment of pregnant pigs. Further, the estrus synchronization treatment was performed so that the estrus of the provisional stomach was delayed one day from that of the egg-collecting pig. The sows to be tested were made pregnant by artificial insemination using boar diluted semen at the time of estrus. Pregnant pigs 21-50 days after pregnancy were used. Six days before the planned nuclear transfer, about 0.2 mg of PGF2α analog cloprosterol (planate, manufactured by Takeda Pharmaceutical Co., Ltd .: 2 ml amount) was injected into the buttocks. After 24 hours, PGF2α analog cloprosterol was similarly injected, and at the same time, eCG (Pimex, Sankyo Pharmaceutical Co., Ltd.) was administered at 1000 IU. 72 hours after administration of PGF2α + eCG, 500 IU of hCG (Puperogen, Sankyo Pharmaceutical Co., Ltd.) was administered. At about 68 hours after hCG administration, ketalal was administered to the abdomen, and then embryo transfer was performed surgically under halothane anesthesia. Embryo transfer was performed by inserting a PP catheter (Fujihira Kogyo Co., Ltd.) into the fallopian tube.

16) Collection of recombinant cloned fetus and cell separation work The identified PCR-positive colonies may contain non-homologous recombinant cells in various proportions. Efficient homologous groups using somatic cell nuclear transfer technology It was anticipated that this could hinder the production of replacement animals. Therefore, in order to obtain a population of only single homologous recombination cells, fetuses were collected on days 39-72 of pregnant sows. The collected fetuses were separated from the cells in the same manner as in 3), and at the same time genomic DNA was collected and subjected to PCR analysis under the same conditions as in 4). Renuclear transfer was performed using fetal cells that were positive in the PCR analysis, and an LDLR gene knockout clone individual was produced by embryo transfer.

17) Southern blot analysis Genomic DNA was extracted from cells or tissues (ears) collected from cloned fetuses or cloned offspring. The cells and minced tissue fragments were treated with lysis buffer (50 mM Tris, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg / ml proteinase K) at 55 ° C. overnight and lysed. . DNA was extracted by ethanol precipitation after treatment with phenol and phenol / chloroform. For Southern blotting, 10 μg of genomic DNA extracted from piglet and fetal cells was cleaved with HindIII or SphI, electrophoresed separately on 0.8% agarose gel, and blotted on Hybond N + membrane. Hybridization was performed using a 5 ′ probe for the membrane blotted with HindIII-treated genomic DNA and a 3 ′ probe for the membrane blotted with genomic DNA treated with SphI. The 5 ′ probe is a PCR method (primer; LDLRint1F1: 5′-ATCCTCCACGATGTTGTTGG -3 ′) containing a part of intron 1 corresponding to the 5 ′ flanking region outside the 5 ′ homologous region, exon 2 and intron 2. And LDLRint2R2: 5′-GGAGGGCCATTAAAGGAAAG-3 ′). In addition, the 3 ′ probe is a PCR method (primer; LDLRint10F3: 5) containing a part of intron 10 corresponding to the 3 ′ adjacent region outside the 3 ′ side homologous region, exon 11 and part of intron 11. '-CCAGTCCTCACACTGGAATG-3' and LDLRint11R3: 5'-GCAAGGCAGCTCAGTTTGGG-3 '). The probe was labeled with [α32-P] dCTP by a random priming method using HighPrime (Roche). The membrane was hybridized using a ³² P-labeled probe, exposed overnight on an Imaging Plate (Fuji Film), and analyzed using a FLA-3000G image analyzer (Fuji Film).

18) PCR determination of the born cloned pig DNA was extracted from tissues (ear, umbilical cord tissue) and blood that could be collected from the born cloned pig, and PCR analysis was performed according to 5).

19) Producing progenies of cloned pigs Most cloned pigs were unable to stand due to symptoms of hyperventilation or hyperthermia, died, and even some surviving individuals could not collect semen and could not obtain progeny Therefore, epididymis spermatozoa were collected from the cadaver at the time of the death of the cloned pig, and progeny were created by in vitro fertilization and artificial insemination. In vitro fertilization was performed according to the method of Kikuchi et al. (Biol Reprod. 2002 Apr; 66 (4): 1033-41). Artificial insemination is performed twice a day in the morning and evening until the end of estrus, using a spiral catheter for porcine artificial insemination using epididymal spermatozoa diluted with modena solution kept at 37 ° C. Conducted for 2-3 days.

20) Determination of homo-type / hetero-type Determination by the PCR method was performed by specifically detecting each of the mutant allele and the wild-type allele. Mutant alleles are detected by the method according to 4), and wild type alleles are detected except that LDLRex3F4 (5'-GCAAGATAGGGGACTTTAGC-3 ') and LDLRex4R1 (5'-GAGTCACAGACAAACTTTGG-3') are used as primers. The same method was used. Homo-type and hetero-type were determined by the combination of alleles detected. The determination by Southern blotting was performed by a method according to 17). Mutant alleles and wild type alleles were discriminated based on the detected band size, and homozygous / heterotyped were determined based on the combination.

21) Blood test (separation of plasma)
Blood was collected from a porcine vein with a heparin blood collection tube, and plasma was separated by centrifugation at 2000 rpm for 15 minutes. The separated plasma was analyzed for the 4-fraction and 20-fraction values of cholesterol and neutral fat by the HPLC method of Okazaki et al. (Arterioscler Thromb Vasc Biol 2005, 25: 578-584).

22) Feed As a feed for pigs, a normal feed (ordinary food) having the following fat content (based on the Japanese standard feed composition table) was used.
3-4 weeks of age: fat content 4.0% (feed name: fried)
5 weeks old to 5 months: Fat content 3.5% (feed name: Hatsutsutsu, Friend U)
After 5 months: Fat content 2.5% (feed name: mother bleed)

<Summary of research results: Homologous recombination cell production test using LDLRex4TV targeting vector>
(1) Cells were collected from porcine L fetal male (68 days old), electroporated with LDLRex4TV targeting vector (FIG. 1) four times, and drug selection with neomycin 400 μg / ml for about one week. After 3 days, the colonies were picked up. The total number of colonies subjected to PCR analysis was 1260 clones, of which 5 clones were positive (Table 1).

(2) Cells were collected from fetal male LW fetus (62 days old), electroporated once with LDLRex4TV targeting vector, drug selection was performed with neomycin at 400 μg / ml for about 1 week, and colonies were observed 2-3 days later. Picked up. The total number of colonies subjected to PCR analysis was 413 clones, of which 1 clone was positive (Table 1).

(3) LDLR gene knockout cell No. obtained in (1). No. 91 was used for nuclear transfer 16 times (3 in vivo mature eggs, 13 in vitro mature eggs), and 3401 divided embryos (170 derived from in vivo mature eggs, 3231 derived from in vitro mature eggs) were obtained (Table 2). ).

(4) As a result of transplanting the divided embryos obtained in (3) to 16 temporary abdomen, 5 became pregnant, and when these temporary abdomen were sacrificed, 6 cloned fetuses were obtained. All obtained fetuses were confirmed to be non-recombinant (Table 2).

(5) No. of LDLR gene knockout cell obtained in (2). 1-16 was used to perform nuclear transfer tests 6 times (1 in vivo ovum and 5 in vitro ova) to obtain 1290 divided embryos (70 in vivo ovum derived, 1220 in vitro ovum derived) (Table 2). ).

(6) As a result of transplanting the divided embryos obtained in (5) to six temporary wombs, two became pregnant and slaughtered those temporary halves, resulting in nine cloned fetuses (FIG. 2A, B). Of the fetuses obtained, 7 were confirmed to be homologous recombinants (Table 2). We succeeded in separating cells in a larger amount than homologous recombinant fetuses.

(7) Furthermore, using the LDLR KO cloned fetal cells derived in (6), seven nuclear transfer / embryo transfer tests were performed. Nuclear transfer was performed using 4742 in vitro matured eggs, 3812 eggs survived (survival rate 80.4%), and 1889 were divided (division rate 49.6%, Table 3). By transplanting 1889 divided embryos into 7 calves, all 7 were pregnant, a total of 38 cloned pigs were born, 29 of which were live births.

(8) As a result of PCR analysis, knockout of LDLR gene was confirmed in all offspring. The born clones had many deaths after birth, and there were many deaths within 1 month and at the age of 4 to 5 months. There were only 2 surviving animals over 7 months of age (Table 4). In many clones, sudden hyperventilation and increased body temperature were observed, and in severe cases, it was impossible to stand and death occurred, which was an unexpected event from other previous studies on the production of knockout pigs. The result of pathological examination was also unclear.

(9) Each cholesterol fraction (VLDL, LDL, HDL) concentration in plasma at the age of 4 to 5 months of the born LDLR gene knockout clone (heterotype) was measured. As a result, it was revealed that the total cholesterol level was more than twice as high as that of the control group, and that the high value in each fraction of VLDL and LDL had an influence as the cause (Table 5 and FIG. 3).

(10) Of the 29 surviving litters, only 2 of the LDLR gene knockout cloned pigs reached sexual maturity after 7 months of age. However, even after 7 months of age, it was not possible to collect injection semen from two animals. One of the two died suddenly at the age of 11 months, and vital sperm was collected from the epididymis tail of the cadaver. Since the remaining one became unable to stand at the age of 12 months, it was euthanized and vital spermatozoa were collected from the epididymis. These epididymal spermatozoa were used for in vitro fertilization and artificial insemination. As a result of transplanting in vitro fertilized embryos obtained by two in vitro fertilizations to four temporary parents, all four became pregnant, and 35 live births and one stillborn baby were born. In addition, 2 animals that had been artificially inseminated became pregnant, and 19 live and 4 live births were obtained (Table 6).

  As a result of examining the genotypes of 51 offspring that led to weaning, it was determined that there were 21 heterozygous LDLR gene knockout pigs (6 males and 15 females) and 30 wild type (16 males and 14 females). (Table 7). After the birth, he grew up steadily without the sudden death of clones. In addition, in some knockout pigs, the plasma cholesterol fractions (VLDL, LDL, HDL) concentrations were measured, and as with F0 clones, the VLDL and LDL concentrations were 2-3 times higher than the wild type. Became clear (data not shown).

(11) Furthermore, four F1 generation males and females were mated to succeed in obtaining 38 F2 generations (Table 8). As a result of examining the genotype of the F2 generation offspring, 12 homozygous LDLR gene knockout pigs (7 males, 5 females), 18 heterozygous (male 9, female 9), 8 wild type (3 males) Female 5) (Table 8).

(12) As a result of measuring 4 fractions of plasma cholesterol (CM, VLDL, LDL, HDL) at 3 and 7 weeks of F2 generation, the homo type was compared to the wild type and hetero type, It was revealed that the concentrations of VLDL and LDL were significantly higher (FIGS. 4A and B). The total cholesterol level in blood at the age of 3 months was 645 mg / dL for homozygous knockout and 137 mg / dL for control (wild type). In addition, the total cholesterol level in blood at the age of 7 months was 598 mg / dL for homozygous knockout and 113 mg / dL for control (wild type).

(13) As a result of measuring the VLDL and LDL concentrations of 4 fractions of plasma cholesterol at 7 weeks of F2 generation, it was revealed that the hetero type was significantly higher than the wild type (FIG. 5B). , P <0.05).

(14) Similarly, as a result of measuring the concentration of 20 fractions of cholesterol in plasma at 3 weeks and 7 weeks of the F2 generation, the homo type is less than the wild type and hetero type, and the G3 fraction is excluded. It was revealed that the concentrations of VLDL in the G4 to G7 fraction and LDL in the G8 to 13 fraction were significantly higher (FIGS. 5A, B, P <0.05).

(15) Further, as a result of measuring the concentration of 20 fractions of cholesterol in plasma at 7 weeks of F2 generation, the heterozygous type showed significant concentrations of LDL in the G8 to G11 and G13 fractions compared to the wild type. (Fig. 5B, P <0.05).

(16) As a result of measuring the HDL value of cholesterol in 4 fractions, the homotype tended to be low, but no significant difference was observed. Furthermore, as a result of detailed analysis at 20 fraction concentrations, homotypes in the G15 fraction had significantly higher HDL values than others (FIGS. 5A, B, P <0.05). On the other hand, in the G17 fraction at the age of 7 weeks, the homotype had a significantly lower HDL value than the others (FIG. 5B, P <0.05).

(17) Total TG (neutral fat: triglyceride) values analyzed in 3 fractions and 7-week-old 4 fractions tended to be higher in homozygous and significantly higher than heterozygous (FIG. 4C, D, P <0.05). In particular, LDL showed significantly higher values for the homotype than the heterotype and the wild type (FIG. 4C, D, P <0.05). On the other hand, in the HDL of 7-week-old neutral fat, the hetero type showed a significantly higher value than the homo type and the wild type (FIG. 4D, P <0.05).
(18) Furthermore, as a result of analyzing TG (neutral fat: triglyceride) of 3 weeks and 7 weeks in detail in 20 fractions, the homozygous type is significant compared to the others in the L10 G10 to G13 fractions. (FIG. 5C, D, P <0.05).

  The present invention can be suitably used in the field of hyperlipidemia research, the field of disease model animals, and the development of therapeutic or preventive drugs for hyperlipidemia.

Claims (6)

In a method in which a pig lacking the function of all or part of the LDLR gene is used as a hyperlipidemia model pig having a HDL cholesterol concentration similar to that of a control and showing hypercholesterolemia even in a normal diet There,
0.8-1.3 times the measured value in wild genotype pigs that did not develop hyperlipidemia and were fed a diet with a fat content of 2-5% to be comparable to the above control Represents
The hypercholesterolemia is state, and are the concentrations of LDL cholesterol and VLDL cholesterol than 2 times the control,
A pig lacking the function of all or part of the LDLR gene enucleated from the collected pig egg, and a part of exon 4 of the LDLR gene was replaced with a resistance gene in the enucleated egg. A somatic cell nuclear transfer cloned animal obtained by injecting a pig somatic cell nucleus or a descendant thereof,
The method according to claim 1, wherein the pig in which the function of all or part of the LDLR gene is deleted has a homozygous mutation in which a part of exon 4 of the LDLR gene is replaced with a resistance gene .   The method according to claim 1, characterized in that the hypercholesterolemia is type IIb hereditary hyperlipidemia. The method according to claim 1 or 2 , wherein the resistance gene is a neomycin resistance gene . The method according to any one of claims 1 to 3, wherein the progeny of the somatic cell nuclear transfer cloned animal is produced by combining in vitro fertilization and artificial insemination techniques. Hyperlipidemia in pig organs or cells derived from pigs lacking all or part of the function of the LDLR gene, in which the concentration of HDL cholesterol is similar to that of the control and hypercholesterolemia is exhibited even in a normal diet A method for use as an organ or cell of a model pig,
0.8-1.3 times the measured value in wild genotype pigs that did not develop hyperlipidemia and were fed a diet with a fat content of 2-5% to be comparable to the above control Represents
The hypercholesterolemia is state, and are the concentrations of LDL cholesterol and VLDL cholesterol than 2 times the control,
A pig lacking the function of all or part of the LDLR gene enucleated from the collected pig egg, and a part of exon 4 of the LDLR gene was replaced with a resistance gene in the enucleated egg. A somatic cell nuclear transfer cloned animal obtained by injecting a pig somatic cell nucleus or a descendant thereof,
The method according to claim 1, wherein the pig in which the function of all or part of the LDLR gene is deleted has a homozygous mutation in which a part of exon 4 of the LDLR gene is replaced with a resistance gene . A method for using a pig lacking the function of all or part of the LDLR gene as a hyperlipidemia model pig comprises a prophylactic or therapeutic agent for hyperlipidemia comprising the following steps (a) to (c): The method according to claim 1, wherein the method is a screening method.
(A) a step of administering a test substance to a pig or a cell derived from the pig, in which the function of all or part of the LDLR gene has been deleted;
(B) a step of examining the concentration of LDL cholesterol or VLDL cholesterol in the test subject in step (a), or the presence or absence of arteriosclerosis;
(C) In the step (b), the test substance is selected as a candidate drug for the prevention or treatment of hyperlipidemia using as an index that the concentration of LDL cholesterol or VLDL cholesterol does not increase or does not develop arteriosclerosis Process;