Hypercholesterolemia / atherosclerosis miniature pig model

Abstract

To provide a miniature swine that is convenient as a model animal of high cholesterol / arteriosclerosis, is small-sized, and can be produced and raised more stably than a cloned miniature swine and whose genotype of an LDLR gene is homo (LDLR- / -).SOLUTION: A non-cloned miniature swine has a genotype of an LDLR gene that is homo (- / -), originates in swines of a plurality of varieties, one of which is a wild-type Berkshire variety, weighs 65 kg or less on average in the case of a male swine that are 270 days old, and weighs 80 kg or less in the case of a female swine.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a non-cloned miniature pig that serves as a model for hypercholesterolemia and arteriosclerosis, in which the low-density lipoprotein receptor (LDLR) gene has been knocked out, a method for producing the same, and uses of the non-cloned miniature pig. [Background technology]

[0002] As lifestyles become more Westernized, cardiovascular diseases are on the rise as one of the three major causes of death in Japan. Among them, the number of patients with ischemic heart disease, such as angina pectoris and myocardial infarction, is increasing year by year. Currently, there are approximately 800,000 patients with ischemic heart disease nationwide, and their medical expenses exceed 700 billion yen. It has been reported that ischemic heart disease occurs due to the progression of arteriosclerosis caused by prolonged hypercholesterolemia. Therefore, in order to treat ischemic heart disease, research and development is required on methods for preventing, diagnosing, and treating hypercholesterolemia and arteriosclerosis. Model animals that develop hypercholesterolemia and arteriosclerosis (hereinafter, these are also referred to as "hypercholesterolemia and arteriosclerosis") are used in these research and development efforts.

[0003] For example, known animal models of hypercholesterolemia and arteriosclerosis include mice with knockouts of the LDLR gene, apolipoprotein gene (ApoE), neuromedin U gene (NMU), etc., which are genes that regulate blood cholesterol levels, and WHHL rabbits, which have been obtained by breeding selection to have a genotype with a LDLR function-decreasing mutation. However, mice and rabbits are small animals with small vascular systems and short life spans, and the compositions of low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol in the blood are significantly different from those of normal humans, and tests using these animals have the problem that they are difficult to use for long-term treatment experiments, surgical treatment tests, development of treatment devices, etc.

[0004] In recent years, pigs have been used for medical research because their anatomical, physiological and biochemical characteristics are similar to those of humans. For example, Patent Document 1 and Non-Patent Documents 1-2 describe a cloned pig model of hypercholesterolemia and arteriosclerosis produced from a large breed of pig, Landrace. However, the cloned pigs described in Patent Document 1 and Non-Patent Documents 1-2 weigh more than 100 kg at 6 months of age and more than 150 kg at 12 months of age, which makes it difficult to raise and manage them in a normal laboratory, and human treatment instruments cannot be used because of their size. As a result, it is difficult to observe the progress of the pigs over a long period of time, making it impossible to observe side effects that occur in the chronic phase after treatment.

[0005] On the other hand, knockout minipigs using small-sized minipigs are also known. Among genes that regulate blood cholesterol levels, deficiency of the LDLR gene can cause familial hypercholesterolemia in humans, increase the risk of coronary artery disease, and ultimately lead to myocardial infarction. Examples of such LDLR gene knockout minipigs are described in Non-Patent Documents 3 to 6. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent No. 6172699 [Non-Patent Document]

[0007] [Non-Patent Document 1] Yuxin Li.et al., J Am Heart Assoc., 5, 1-13 (2016) [Non-Patent Document 2] Manabu Ogita et al., 2016 PLoS One., 11, e0163055 (2016) [Non-Patent Document 3] Lei Huang et al. Oncotarget., 8, 37751-37760 (2017) [Non-Patent Document 4] Bryan T. Davis et al., PLoS One., 9(4), e93457 (2014) [Non-Patent Document 5] Chidozie Amuzie et al., Toxicol Pathol., 44(3), 442-449 (2016) [Non-Patent Document 6] Amy C. Burke et al., Arterioscler. Thromb. Vasc. Biol., 38, 1178-1190 (2018) [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] The LDLR gene knockout minipigs described in Non-Patent Documents 3 to 6 are cloned minipigs or their progeny. In general, somatic cell cloned animals and their progeny have low conception and parturition rates and high mortality rates around birth or before weaning, so there is a high probability that the cloned minipigs described in Non-Patent Documents 3 to 6 cannot be stably produced and raised. Even if they could be produced, the average number of offspring born to minipigs is small, about 5, and coupled with the high mortality rate, the number of cloned minipigs that will grow to the point where they can reproduce may be only one to a few.

[0009] Therefore, the objective of the present invention is to provide a miniature pig with a homozygous genotype (LDLR- / -) for the LDLR gene, which is useful as a model animal for hypercholesterolemia and arteriosclerosis, yet is small and can be produced and raised more stably than cloned miniature pigs. [Means for solving the problem]

[0010] In order to solve the above problems, the inventors aimed to miniaturize the pig by crossbreeding a nuclear transfer somatic cell cloned pig (LDLR+ / -) with a heterozygous knockout of the LDLR gene, which was produced using Landrace (L), a breed widely distributed for meat as described in Patent Document 1, and a three-way crossbreed pig, LWD (Landrace x Large Yorkshire (W) x Duroc (D)), with Sakura Koyuki, a minipig (LDLR+ / +) developed at the Livestock Breeding Center.

[0011] However, the difference in size between cloned pigs and minipigs was about three times that of their predecessors, making natural mating impossible. When they tried to use artificial insemination, they ran into great difficulties because there was no established method for collecting semen from minipigs.

[0012] Therefore, the inventors, through repeated trial and error, created a small dummy suitable for the physique of a miniature pig, established a method for collecting semen using the dummy by hand pressure after training, and succeeded in producing piglets (S line F1 generation) by mating the cloned pig with the miniature pig by artificial insemination. However, the S line F1 generation produced had a high immediate postnatal mortality rate and a low growth rate until weaning, and many individuals were unable to grow.

[0013] Despite the above problems, we selected pigs with heterozygous genotype (LDLR+ / -) from the S line F1 generation and crossed them with each other to select for body shape and genes, in an attempt to obtain individuals with homozygous genotype (LDLR- / -) for the LDLR gene. As a result, we succeeded in obtaining individuals with homozygous genotype (LDLR- / -) in the F3 generation. The homozygous (LDLR- / -) minipigs produced were small, weighing about 50 kg at 9 months of age, and showed pathological symptoms of hypercholesterolemia and arteriosclerosis that are highly similar to those in humans. However, the litter size was small, many died immediately after birth, the weaning rate on the 35th day after birth was very low, and the pigs had low disease resistance, with sudden death and skin diseases occurring frequently even in the rearing pigs. In addition, the piglets were unable to suckle due to enlarged nipples. Due to these problems, it was extremely difficult to maintain and manage the homozygous (LDLR- / -) minipigs of the S line F3 generation. In addition, all generations of the S line had problems such as sudden death between 3 and 6 months of age, onset of systemic skin disease, loss of energy immediately after feeding a high-fat diet, frequent loss of appetite, and mass death, and many individuals could not be used for the intended experiments, making them unsuitable for maintaining and continuously supplying pig groups. The underlying cause of this is that since cloned pigs were produced from recombinant cells of the same individual and the minipigs were also of one breed, in order to obtain individuals with a homozygous LDLR gene genotype (LDLR- / -), inbreeding may have progressed early, leading to a decrease in reproductive ability and disease resistance.

[0014] Therefore, the inventors of the present invention have been working on further research and development, and as a result of intensive investigation, have come up with the idea of ​​crossbreeding homozygous (LDLR- / -) pigs of the S lineage F3 generation with British Berkshire pigs. Berkshire pigs are intermediate in size between large pigs such as Landrace and Large Yorkshire and minipigs. In addition, the Berkshire breed is distantly related to Landrace, Large Yorkshire and minipigs from the pedigree tree, yet the blood ties within the lineage are highly maintained, and it is possible to maintain and supply them for a long time by continuously using them for blood ties renewal. Furthermore, by crossbreeding with another breed, the hybrid vigor effect (heterosis effect) works, and it is expected that the resulting progeny minipigs will be robust and have an increased number of offspring.

[0015] Based on the above idea, S line F3 generation homozygous (LDLR- / -) pigs, which were produced by crossing a large breed with a miniature pig, were crossed with Berkshires (LDLR+ / +). The resulting SB line F1 generation had a large body size, and the genotype was heterogeneous. Therefore, the SB line F1 generation was selected based on body size, and the SB line F2 generation was obtained by crossing the SB line F1 generation pigs with each other or the SB line F1 generation pigs with miniature pigs.

[0016] Among the SB strain F2 generation, there were homozygous (LDLR- / -) strains, and the SB strain F2 generation was further selected based on genotype and body shape, and the SB strain F2 generation homozygous (LDLR- / -) strains were crossed with each other or with homozygous and heterozygous (LDLR+ / -) strains to obtain the SB strain F3 generation.

[0017] Furthermore, based on the genotype and body shape, we succeeded in obtaining all homozygous SB lineage F4 generation (LDLR- / -) pigs by crossing small SB lineage F3 generation homozygous (LDLR- / -) pigs with each other.

[0018] Surprisingly, the obtained SB lineage F4 generation homozygous (LDLR- / -) pigs are small, have many offspring, have high survival rates and weaning rates immediately after birth, and are robust, making them stable to produce and raise, and excellent as model animals for hypercholesterolemia and arteriosclerosis. As mentioned above, somatic cell cloning techniques such as nuclear transfer were not used to obtain the SB lineage F1 to F4 generations, and the obtained SB lineage F4 generation homozygous (LDLR- / -) pigs are non-cloned minipigs.

[0019] Based on the above findings and success stories, the present inventors have finally succeeded in creating non-cloned mini-pigs that can be used as model animals for hypercholesterolemia and arteriosclerosis, a method for producing non-cloned mini-pigs, and a method for using non-cloned mini-pigs, which can solve the problems of the present invention. The present invention has been completed based on findings and success stories first discovered by the present inventors.

[0020] Therefore, according to the present invention, there are provided the following aspects of a non-cloned mini-pig, a method for producing a non-cloned mini-pig, a method for screening a candidate substance, and organs and cells. [1] The genotype of the LDLR gene is homozygous (- / -); derived from multiple breeds of pigs, one of which is a wild-type Berkshire; and The average body weight at 270 days is 65 kg or less for males and 80 kg or less for females. Non-cloned mini pigs. [2] The non-cloned mini-pig described in [1] has a conception rate of 80% or more, a birth rate of 80% or more, an average litter size of 8 or more, and / or an average pre-weaning mortality rate of 20% or less. [3] The non-cloned minipig according to any one of [1] to [2], wherein the non-cloned minipig has an average nipple height of 15 mm or less and / or an average nipple diameter of 12 mm or less. [4] The non-cloned mini-pig described in any one of [1] to [3], which is used as a model animal for at least one disease selected from the group consisting of hypercholesterolemia and arteriosclerosis. [5] The non-cloned miniature pig described in [4], wherein the hypercholesterolemia is type IIb hereditary hyperlipidemia. [6] A non-cloned mini-pig described in any one of [1] to [5], wherein the non-cloned mini-pig has a total cholesterol concentration in venous plasma of 220 mg / dl or more and / or an LDL cholesterol concentration in venous plasma of 140 mg / dl or more when ingesting a feed containing 2% to 5% by mass of fat for two months. [7] A step of crossbreeding a pig having a heterozygous (+ / -) or homozygous (- / -) genotype of the LDLR gene and derived from a breed including the Landrace breed with a wild-type miniature pig having a wild (+ / +) genotype of the LDLR gene by artificial insemination, and then using the resulting pig for subculture to obtain a first miniature pig having a homozygous (- / -) genotype of the LDLR gene; A step of crossbreeding the first miniature pig with a Berkshire pig by artificial insemination, and then using the resulting pig for subculture to obtain a non-cloned miniature pig having a homozygous (- / -) genotype of the LDLR gene. A method for producing a non-cloned miniature pig according to any one of [1] to [6], comprising: [8] A step of administering a test substance to a non-cloned miniature pig according to any one of [1] to [6] or a cell derived from the non-cloned miniature pig as a test subject, and measuring the concentration of at least one type of cholesterol selected from the group consisting of LDL cholesterol and VLDL cholesterol or the degree of arteriosclerosis in the test subject; screening the test substance as a candidate substance for preventing or treating hypercholesterolemia or arteriosclerosis based on whether the cholesterol concentration or the degree of arteriosclerosis is the same or lower than that of a control; A method for screening a candidate substance for the prevention or treatment of hypercholesterolemia or arteriosclerosis, comprising: [9] The screening method described in [8], wherein the control is at least one type of control selected from the group consisting of the concentration and level before administration of the test substance, and the concentration and level when the test subject is a wild-type pig and cells derived from a wild-type pig.

[10] An organ or cell derived from a non-cloned miniature pig described in any one of [1] to [6]. Effect of the Invention

[0021] According to the present invention, it is possible to provide a non-cloned miniature pig with a homozygous genotype (LDLR- / -) for the LDLR gene, which is small while being useful as a model animal for hypercholesterolemia and arteriosclerosis. The non-cloned miniature pig of one embodiment of the present invention has a high conception rate and birth rate despite its small size, a larger average litter size than miniature pigs, and the small size of its nipples makes it easy to suckle piglets, allowing for stable production and growth. Furthermore, the non-cloned miniature pig of one embodiment of the present invention is relatively robust, and can be used for long-term drug administration tests, etc. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a graph showing the stillbirth and postnatal mortality rates in each generation of the SB and S strains, as described in the Examples below. [Diagram 2] FIG. 2 is a diagram showing the mortality rates of the SB and S strains in each generation, as described in the Examples below. [Diagram 3] FIG. 3 shows the genotypes of the SB and S strains in each generation, as described in the Examples below. [Figure 4] FIG. 4 shows the results of measuring the genotype of offspring derived from frozen semen, as described in the Examples below. [Diagram 5] FIG. 5 shows the results of measuring blood lipid concentrations in SB lineage F3 generation homozygous (LDLR- / -) miniature pigs and homozygous (LDLR- / -) pigs described in Patent Document 1, as described in the Examples below. [Figure 6] FIG. 6 shows the results of measuring blood lipid concentrations in SB strain F3 generation homozygous (LDLR- / -) miniature pigs after feeding a high-fat diet, as described in the Examples below. [Figure 7] FIG. 7 shows photographs of coronary angiography (B) and IVUS (C to E) as described in the Examples below. [Figure 8] 8 is a set of photographs obtained by microscopic observation of the coronary artery and aorta stained with HE as described in the Examples below. The photograph on the left shows the aorta, and the photograph on the right shows the coronary artery. [Figure 9] 9 is a set of photographs obtained by pathological staining and microscopic observation 12 months after placement of a DES or BVS, as described in the Examples below. The photographs on the left show the conditions for DES placement, and the photographs on the right show the conditions for BVS placement. [Figure 10] 10 is a set of photographs obtained by pathological staining and microscopic observation one month after placement of the second DES, as described in the Examples below. The photograph on the left shows an LWD three-way crossbred pig, and the photograph on the right shows an SB homozygous (LDLR- / -) miniature pig. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The non-cloned mini-pig, the method for producing the non-cloned mini-pig, and the method for screening a candidate substance, which are one embodiment of the present invention, are explained in detail below, but the present invention can take various forms as long as it achieves its object.

[0024] Unless otherwise specified, each term in this specification is used in the sense commonly used by those skilled in the art and should not be interpreted as having an unduly limiting meaning. In addition, the speculations and theories made in this specification are based on the inventors' past knowledge and experience, and therefore the present invention is not limited solely to such speculations and theories.

[0025] A "wild-type" pig means a pig that has not been genetically engineered. The genotype of the LDLR gene in a "wild-type" pig is wild (+ / +). A pig that has been genetically engineered at a locus other than the LDLR locus by genetic engineering or genome editing technology may be used as a wild-type pig, provided that the phenotype, such as body size, growth ability, and reproductive ability, is not different from that of a wild-type pig. "And / or" means any one or any or all combinations of two or more of the associated listed items. "Comprising" means that elements other than the elements explicitly stated as being included can be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." In other words, "comprising" can mean including the elements explicitly stated and any one or more elements, consisting of the elements explicitly stated, or consisting essentially of the elements explicitly stated. Elements include limitations such as ingredients, steps, conditions, parameters, etc.

[0026] [1. Non-cloned mini pigs] The non-cloned miniature pigs of one embodiment of the present invention are small-sized miniature pigs that are homozygous (- / -) for the LDLR gene and originate from multiple breeds including the Berkshire breed.

[0027] A specific embodiment of the non-cloned miniature pig of the present invention is the SB strain miniature pig described in the Examples below. The outline of this SB strain homozygous (LDLR- / -) miniature pig is described below.

[0028] The SB line homozygous (LDLR- / -) miniature pigs created by the present inventors and described in the Examples below are model miniature pigs of hypercholesterolemia and arteriosclerosis, which were produced by applying the hyperlipidemic model pigs described in Patent Document 1. The hyperlipidemic model pigs described in Patent Document 1 weigh more than 100 kg at 6 months of age and more than 150 kg at 9 months of age, making them difficult to handle for long periods in experiments. Therefore, we attempted to reduce the size of the hyperlipidemic model pigs described in Patent Document 1 by crossbreeding them with a miniature pig (Sakura Koyuki) developed at the Livestock Breeding Center, and produced the hyperlipidemic model miniature pig line S. However, although the S line miniature pigs were reduced in size, they were difficult to produce and maintain efficiently due to problems such as inconsistency in physique during the production process, reduced reproductive ability due to inbreeding degeneration of the produced pigs, reduced rearing rate, and inferior robustness compared to normal pigs, making them unsuitable as experimental animals. Therefore, by crossing the S strain miniature pig with a medium-sized British Berkshire breed and repeatedly selecting genes and body shape, we succeeded in homozygous breeding and producing the homozygous (LDLR- / -) miniature pig strain SB, a high cholesterol / atherosclerosis model that weighed less than 50 kg at 6 months of age and less than 80 kg at 9 months of age.

[0029] The SB homozygous (LDLR- / -) miniature pigs are characterized by homozygous genes, small body size, improved fertility, improved rearing rate, and robustness comparable to that of normal pigs. Furthermore, the SB homozygous (LDLR- / -) miniature pigs exhibited symptoms of hypercholesterolemia even when fed a normal diet, and the composition of plasma cholesterol (VLDL: very low density lipoprotein, LDL, and HDL cholesterol) fully reproduced human familial hyperlipidemia. In addition, the SB homozygous (LDLR- / -) miniature pigs exhibited arteriosclerotic lesions such as unstable atherosclerosis in coronary arteries similar to those in humans, and the treatment methods and chronic side effects after treatment were similar to those in humans. Therefore, the SB homozygous (LDLR- / -) miniature pigs are more excellent model animals for diagnosing hypercholesterolemia and arteriosclerosis in humans, screening for therapeutic agents, preventive agents, chronic side effects after treatment, and elucidation of the mechanism of their occurrence, than the hyperlipidemic pig model described in Patent Document 1.

[0030] The non-cloned mini pig of one embodiment of the present invention is characterized in that it is not a cloned mini pig. That is, the non-cloned mini pig of one embodiment of the present invention is not a cloned mini pig itself produced by somatic cell cloning technology by nuclear transfer. The non-cloned mini pig of one embodiment of the present invention is derived from a wild-type Berkshire breed that has not been produced by somatic cell cloning technology, and can be a non-cloned mini pig in that sense.

[0031] The breed of origin of the non-cloned miniature pig of one embodiment of the present invention is not particularly limited as long as it includes wild-type Berkshire, but in order to maintain small size over generations, it is preferable to further include miniature pigs such as Sakurakoyuki, and more preferably to maintain robustness over generations, it is more preferable to further include Landrace, Large Yorkshire, and Duroc. Note that a breed such as wild-type Berkshire may be one breed included in a crossbreed. The SB line described in the examples below is derived from Landrace, Large Yorkshire, Duroc, Sakurakoyuki, and Berkshire. The S line described in the examples below is derived from Landrace, Large Yorkshire, Duroc, and Sakurakoyuki.

[0032] The non-cloned mini pig of one embodiment of the present invention is smaller in body size and weight than Landrace pigs raised for meat and wild-type Berkshire pigs used for breeding. The non-cloned mini pig of one embodiment of the present invention has an average body weight of 65 kg or less at 270 days old when it is male, and is preferably 60 kg or less, more preferably 50 kg or less, from the viewpoint that the smaller the body size, the easier it is to raise and use in various tests. The non-cloned mini pig of one embodiment of the present invention has an average body weight of 80 kg or less at 270 days old when it is female, and is preferably 78 kg or less, more preferably 70 kg or less, from the viewpoint that the smaller the body size, the easier it is to raise and use in various tests.

[0033] In one embodiment of the present invention, the non-cloned miniature pig preferably has a small weight at birth and at weaning (35 days old) from the viewpoint of ease of parturition and rearing, in addition to the weight at the day used for various tests. In one embodiment of the present invention, the non-cloned miniature pig preferably has an average birth weight of less than 1 kg, more preferably 0.7 kg or less, and even more preferably 0.6 kg or less. In addition, in one embodiment of the present invention, the non-cloned miniature pig preferably has an average weaning weight of 7.5 kg or less, more preferably 6.0 kg or less, and even more preferably 5.5 kg or less.

[0034] According to the inventors' investigation, the cloned pigs and their progeny pigs (hereinafter collectively referred to as "clone pigs") produced using Landrace pigs and three-way crossbred pigs (Landrace x Large Yorkshire x Duroc), as well as the first non-clone mini pigs and their progeny pigs (hereinafter collectively referred to as "first cloned mini pigs") produced by crossbreeding them with the mini pig Sakura Koyuki by artificial insemination, had low conception and parturition rates and low average litter size. However, by crossbreeding the first non-clone mini pig with a Berkshire pig by artificial insemination to produce a second non-clone mini pig, further artificially inseminating the second non-clone mini pigs with each other, or the second non-clone mini pig with the mini pig Sakura Koyuki, and continuing to artificially inseminate the progeny pigs with each other, progeny pigs of the second non-clone mini pig with high conception and parturition rates and high average litter size were obtained.

[0035] In view of the above findings, it is preferable that the non-cloned mini pig of one embodiment of the present invention has a higher conception rate, a higher birth rate, and a larger average litter size than the cloned pig and the first cloned mini pig by including a wild-type Berkshire breed in the breed from which it is derived. Therefore, the non-cloned mini pig of one embodiment of the present invention has a conception rate of preferably 80% or more, more preferably 90% or more; a birth rate of preferably 80% or more, more preferably 85% or more; and / or an average litter size of preferably 8 or more, more preferably 9 or more, even more preferably 9.5 or more.

[0036] As described in the Examples below, the S lineage F1 to F3 generation minipigs corresponding to the first non-cloned minipigs had a high pre-weaning mortality rate of 40% or more. In contrast, the SB lineage F1 to F4 generation minipigs corresponding to the progeny pigs of the second non-cloned minipigs had a pre-weaning mortality rate of less than 40%. Taking these circumstances into consideration, the non-cloned minipigs of one embodiment of the present invention preferably have a pre-weaning mortality rate of less than 40%, more preferably 30% or less, and even more preferably 20% or less.

[0037] As described in the Examples below, enlargement of nipples was observed in the S lineage F3 generation minipigs, but not in the SB lineage F3 generation minipigs. Therefore, the non-cloned minipigs of one embodiment of the present invention preferably have a smaller average nipple height than the first non-cloned minipigs, more preferably 15 mm or less; and preferably have a smaller average nipple diameter than the first non-cloned minipigs, more preferably 12 mm or less.

[0038] For non-cloned minipigs of one embodiment of the present invention, the average body weight at 270 days of age, conception rate, birth rate, average number of offspring born, average pre-weaning mortality rate, average teat height and average teat diameter may be measured by the methods described in the Examples below.

[0039] In one embodiment of the present invention, the genotype of the LDLR gene is homozygous (- / -). In theory, when the genotype of the LDLR gene in a living individual is homozygous (- / -), a normally functioning LDLR gene expression product is not obtained, and symptoms or abnormalities associated with the lack of expression of the LDLR gene appear in the individual. Therefore, the non-cloned miniature pig of one embodiment of the present invention is preferably used as a model animal for symptoms or abnormalities that appear when the genotype of the LDLR gene is homozygous (- / -), and more preferably used as a model animal for hypercholesterolemia, arteriosclerosis, or both diseases.

[0040] In this specification, "hypercholesterolemia" refers to a disease commonly used in the medical community. For example, hypercholesterolemia means that the cholesterol level in the blood is abnormal, specifically, that the total cholesterol concentration in plasma when blood is drawn in a fasting state is 220 mg / dl or more. In this specification, hypercholesterolemia is essentially the same as or includes dyslipidemia and hyperlipidemia, and no distinction is made between them. That is, the non-cloned miniature pig of one embodiment of the present invention may be used as a model animal for dyslipidemia and hyperlipidemia.

[0041] Human hyperlipidemia can be classified into types I, IIa, IIb, III, IV, and V based on the amount of lipid components in the blood. Among these, type IIb is hyperlipidemia in which the VLDL cholesterol concentration and LDL cholesterol concentration are higher than those of healthy subjects, and the HDL cholesterol concentration is similar to that of healthy subjects. The non-cloned miniature pig of one embodiment of the present invention tends to have higher VLDL cholesterol concentration and LDL cholesterol concentration and a similar HDL cholesterol concentration compared to pigs (controls) whose genotype of the LDLR gene is wild (LDLR+ / +), and can show a blood lipid component composition similar to that of IIb hereditary hyperlipidemia. Therefore, the non-cloned miniature pig of one embodiment of the present invention is preferably used as a model animal for type IIb hereditary hyperlipidemia.

[0042] In this specification, "arteriosclerosis" refers to a disease as commonly used in the medical community, for example, arteriosclerosis refers to a disease in which plaque forms in the arteries.

[0043] The concentrations of HDL cholesterol, LDL cholesterol and VLDL cholesterol can be measured as the HDL, LDL and VLDL concentrations in the cholesterol fraction of a blood-derived sample such as plasma, serum, etc. Similarly, the concentrations of HDL triglyceride, LDL triglyceride and VLDL triglyceride can be measured as the HDL, LDL and VLDL concentrations in the triglyceride (neutral fat) fraction of a blood-derived sample such as plasma, serum, etc.

[0044] For example, a method for quantifying VLDL cholesterol, LDL cholesterol, and HDL cholesterol may include, for example, a method in which a serum sample is first fractionated into each cholesterol, and each fraction is then quantified by an enzymatic method using an enzymatic method kit such as "Determiner L TCII," "Determiner TC-555" (manufactured by Kyowa Medex Co., Ltd.), or "Aqua Auto Kainos T-CHO Reagent" (manufactured by Kainos Co., Ltd.). In addition, a method for fractionating each cholesterol may include, but is not limited to, a method of analyzing lipoproteins in serum by high performance liquid chromatography (HPLC) using a gel filtration column such as "TSKgel LipopropakXL, TSKgel Lipopropak" (manufactured by Tosoh Corporation).

[0045] The non-cloned miniature pig of one embodiment of the present invention is not particularly limited in terms of blood lipid component concentrations, such as total cholesterol, total triglycerides, HDL cholesterol, LDL cholesterol, and VLDL cholesterol, as these concentrations vary depending on the age, rearing environment, type of feed, etc.; however, for example, in order to be used as a model experimental animal for hypercholesterolemia and / or arteriosclerosis, when fed a feed containing 2% to 5% by mass of fat for two months, the total cholesterol concentration in venous plasma is preferably 220 mg / dl or more, more preferably 300 mg / dl or more; and the LDL cholesterol concentration in venous plasma is preferably 140 mg / dl or more, more preferably 200 mg / dl or more.

[0046] In this specification, a feed with a fat content of 2% to 5% by mass is also called a normal diet. The normal diet may be a commercially available product described in the Examples below. A feed with a higher fat content than a normal diet is also called a high-fat diet. A high-fat diet can be prepared by mixing a fat-containing material such as lard, high-fat edible oil, or oil and fat into a normal diet. The fat content in a high-fat diet may be set to 10% to 50% by mass. Generally, a wild-type pig (LDLR+ / +) fed a normal diet has a total cholesterol level in the blood of 50 mg / dl to 100 mg / dl, whereas a wild-type pig (LDLR+ / +) fed a high-fat diet has a total cholesterol level in the blood of 500 mg / dl to 800 mg / dl, and develops hyperlipidemia.

[0047] [2. How to create non-cloned minipigs] The method for producing a non-cloned mini-pig of one embodiment of the present invention is not particularly limited, so long as it uses at least a wild-type Berkshire breed, the genotype of the LDLR gene of the resulting non-cloned mini-pig is homozygous (- / -), and the average body weight at 270 days of age is the specified body weight.

[0048] Theoretically, it is possible to create minipigs with homozygous (- / -) genotypes of the LDLR gene by utilizing genetic engineering and somatic cell cloning technology using nuclear transfer. However, because minipigs are produced by inbreeding, cloned minipigs have extremely poor reproductive ability and robustness, and it is extremely difficult for them to give birth and grow, making it almost impossible to create progeny of minipigs.

[0049] In view of the above circumstances, it is preferable not to use cloned minipigs to obtain the non-cloned minipigs of one embodiment of the present invention. For this purpose, a pig having a heterozygous (+ / -) or homozygous (- / -) genotype of the LDLR gene is crossed with a wild-type minipig by artificial insemination, and the resulting pig is used for subculture to obtain a first minipig having a homozygous (- / -) genotype of the LDLR gene. However, the first minipigs thus obtained are still inferior in reproductive ability and robustness, and are difficult to use as model animals for hypercholesterolemia and arteriosclerosis in long-term tests. In addition, the first minipigs have physical abnormalities such as enlarged nipples, and are difficult to grow.

[0050] However, by crossing the first mini-pig with a Berkshire pig by artificial insemination and then using the resulting pig to pass on the offspring, it is possible to obtain a mini-pig that has a homozygous (- / -) genotype for the LDLR gene and is approximately the same size as the first mini-pig, but has excellent reproductive ability and robustness, produces large numbers of offspring, and does not show enlarged nipples.

[0051] The non-cloned miniature pig of one embodiment of the present invention is preferably produced based on the above findings. That is, as another embodiment of the present invention, a method for producing a non-cloned miniature pig of one embodiment of the present invention includes the following steps: A step of crossbreeding a pig having a heterozygous (+ / -) or homozygous (- / -) genotype of the LDLR gene and derived from a breed including the Landrace breed with a wild-type miniature pig having a wild (+ / +) genotype of the LDLR gene by artificial insemination, and further subculturing the pig obtained, thereby obtaining a first miniature pig having a homozygous (- / -) genotype of the LDLR gene; and A step of crossbreeding the first miniature pig with a Berkshire pig by artificial insemination, and then using the resulting pig for passage to obtain a non-cloned miniature pig having a homozygous (- / -) genotype for the LDLR gene.

[0052] Pigs having a heterozygous (+ / -) or homozygous (- / -) genotype of the LDLR gene and derived from a breed including Landrace are not particularly limited, but examples include pigs having a heterozygous (LDLR+ / -) or homozygous (- / -) genotype of the LDLR gene of the F2 generation LDLR-KO pig described in Patent Document 1, of which heterozygous (LDLR+ / -) pigs are preferred because of their relative robustness.

[0053] The pigs having a heterozygous (+ / -) genotype of the LDLR gene of the LDLR-KO pig F2 generation described in Patent Document 1 can be obtained by a method using the somatic cell cloning technique by nuclear transfer described in Patent Document 1. For example, the collected pig eggs are enucleated, and the enucleated eggs are injected with pig somatic cell nuclei having genomic DNA in which a part or all of the LDLR gene has been modified or deleted, and the resulting nuclear-transferred eggs are implanted in the uterus of a parent female pig, and the pig is allowed to become pregnant and give birth, thereby producing a somatic cell nuclear transfer cloned pig. As described in Patent Document 1, the pig LDLR gene is registered in databases such as NCBI (National Center for Biotechnology Information) under Gene ID: 396801, and the DNA sequence can be obtained from GenBank under accession number NC_010444.3, version GI: 347618792.

[0054] Examples of wild-type minipigs having a wild (+ / +) genotype of the LDLR gene include, but are not limited to, Sakura Koyuki minipigs available from the National Livestock Breeding Center.

[0055] Since pigs derived from breeds including Landrace are large and wild-type minipigs are small, taking into consideration the ease of childbirth, it is preferable to use pigs with a heterozygous (+ / -) or homozygous (- / -) genotype of the LDLR gene and derived from a breed including Landrace as breeding female pigs, and wild-type minipigs with a wild (+ / +) genotype of the LDLR gene as breeding male pigs.

[0056] The method of mating the breeding sow and the breeding male pig by artificial insemination is not particularly limited, and mating by artificial insemination of normal pigs can be adopted. However, since it is difficult to collect semen from a small miniature pig to be used as a breeding male pig, it is preferable to collect semen using a small dummy described in the examples below.

[0057] Pigs of the F2 generation are obtained by crossbreeding pigs (F1 generation) obtained by artificial insemination of multiple breeding female pigs and multiple breeding male pigs. Also, pigs of the F2 generation are crossbreeded with each other, or pigs of the F2 generation and pigs of the F1 generation are crossbreeded to obtain pigs of the F3 generation. By passaging in this manner, a first miniature pig having a homozygous (- / -) genotype of the LDLR gene is obtained. It is preferable to select a pig of relatively small size as the miniature pig to be used for passaging. It is preferable that the first miniature pig having a homozygous (- / -) genotype of the LDLR gene is an F3 generation miniature pig.

[0058] The first miniature pig is mated with a wild-type Berkshire pig by artificial insemination. The method for obtaining the wild-type Berkshire pig is not particularly limited, but it can be obtained, for example, from the Saitama Prefectural Agricultural Technology Research Center. The wild-type Berkshire pig has a size intermediate between a large breed such as a Landrace and a miniature pig. The sex of the first miniature pig and the wild-type Berkshire pig is not particularly limited, but since semen can be collected using a small dummy, it is preferable that the first miniature pig is a breeding male miniature pig and the Berkshire pig is a breeding female pig.

[0059] A miniature pig (F1 generation) obtained by crossbreeding a plurality of breeding female pigs and a plurality of breeding male miniature pigs by artificial insemination is crossed with each other, or a miniature pig of the F1 generation is crossed with a wild-type miniature pig to obtain a miniature pig of the F2 generation, and then the miniature pigs of the F2 generation are crossed with each other to obtain a miniature pig of the F3 generation, and then the miniature pigs of the F3 generation are crossed with each other to obtain a miniature pig of the F4 generation. By passing in this manner, a non-cloned miniature pig having a homozygous (- / -) genotype of the LDLR gene can be obtained. It is preferable to select a miniature pig with a relatively small size for use in the passaging. For example, when a miniature pig having a homozygous (- / -) genotype of the LDLR gene is used as an F3 generation miniature pig to obtain a F4 generation miniature pig, all the miniature pigs of the F4 generation are non-cloned miniature pigs having a homozygous (- / -) genotype of the LDLR gene.

[0060] The non-cloned minipigs thus obtained are small in size, even though they are derived from the medium-sized Berkshire breed. Furthermore, compared with the first minipigs or wild-type minipigs, the non-cloned minipigs tend to have higher conception and parturition rates, larger average litter size, and lower average pre-weaning mortality. In addition, in the non-cloned minipigs, as a rule, nipple enlargement as seen in the first minipigs is not observed. Nevertheless, since the genotype of the LDLR gene is homozygous (- / -), the non-cloned minipigs develop symptoms of hypercholesterolemia and arteriosclerosis even when raised on a normal diet, just like the pigs of the LDLR-KO pig F2 generation described in Patent Document 1, which have a homozygous (- / -) genotype of the LDLR gene, and can be used as a model animal for the disease.

[0061] [3. How to use non-cloned minipigs] The method of using the non-cloned miniature pig of one embodiment of the present invention is not particularly limited, and it is preferable to use it as a model animal for hypercholesterolemia and arteriosclerosis, and secondary diseases caused by these diseases, such as myocardial infarction and cerebral infarction. Specifically, the non-cloned miniature pig of one embodiment of the present invention can be used as a model animal for hypercholesterolemia and arteriosclerosis under normal diet feeding, to determine diseases such as hypercholesterolemia, arteriosclerosis, myocardial infarction, and cerebral infarction (hereinafter, these are also collectively referred to as "diseases such as hypercholesterolemia and arteriosclerosis"), to develop treatment techniques and devices for diseases such as hypercholesterolemia and arteriosclerosis, and to use as a practice rig for learning treatment techniques for diseases such as hypercholesterolemia and arteriosclerosis.

[0062] One embodiment of the non-cloned miniature pigs of the present invention tend to develop hypercholesterolemia and arteriosclerosis even when fed a normal diet, but they can be fed a high-fat diet and maintained in a conditioned environment to age them in order to intensify the symptoms of these diseases or induce secondary diseases such as myocardial infarction and cerebral infarction.

[0063] Examples of therapeutic techniques for diseases such as hypercholesterolemia and arteriosclerosis include, but are not limited to, stent placement, bypass surgery, catheter surgery, carotid endarterectomy (CEA), etc. Examples of therapeutic devices for diseases such as hypercholesterolemia and arteriosclerosis include, but are not limited to, stents, catheters, etc.

[0064] Using non-cloned minipigs and their blood, blood vessels and tissues, it is possible to practice judging the presence or absence of diseases such as hypercholesterolemia, arteriosclerosis, etc. For example, it is possible to practice judging the presence or absence of diseases such as hypercholesterolemia, arteriosclerosis, etc. from the measurement results of the heart rate, pulse rate, blood pressure, etc., blood test results such as blood glucose level, red blood cell count, white blood cell count, blood LDL cholesterol level, blood HDL cholesterol level, and blood VLDL cholesterol level, and test results such as ultrasound examination (echo) and magnetic resonance imaging (MRI).

[0065] For body fluids such as blood, blood vessels, heart, and other tissues collected from non-cloned minipigs, blood tests, dissections, histochemical staining, etc. can be performed to practice diagnosing diseases such as hypercholesterolemia and arteriosclerosis. For example, non-cloned minipigs that have developed diseases such as arteriosclerosis, myocardial infarction, and cerebral infarction can be used to practice diagnosing these diseases in humans using individual pigs, blood vessels with atheroma and tissues containing said blood vessels, and hearts with myocardial infarction.

[0066] Furthermore, the non-cloned miniature pig of one embodiment of the present invention can be used as an animal for evaluating drug efficacy. A candidate drug for treating diseases such as hypercholesterolemia and arteriosclerosis can be administered to non-cloned miniature pigs fed a normal diet or a high-fat diet, and the efficacy and side effects of the candidate drug can be examined using as indicators the improvement of blood cholesterol levels, symptoms, or abnormalities, or the absence of aggravation of these.

[0067] The non-cloned mini-pigs of one embodiment of the present invention, even when fed a normal diet, tend to have higher LDL cholesterol and VLDL cholesterol concentrations and similar HDL cholesterol concentrations compared to wild-type pigs, and may exhibit hypercholesterolemia and arteriosclerosis. By utilizing such properties of the non-cloned mini-pigs of one embodiment of the present invention, organs or cells derived from the non-cloned mini-pigs can be used for screening of candidate substances for preventing and treating hypercholesterolemia and arteriosclerosis. Another embodiment of the present invention is organs and cells derived from the non-cloned mini-pigs of one embodiment of the present invention.

[0068] The organs and cells of one embodiment of the present invention may be any biological sample derived from a non-cloned mini-pig of one embodiment of the present invention, and examples of such biological tissues include those in the head, neck, chest, abdomen, legs, etc., such as organs, bones, body fluids, and cells. Examples of such cells include somatic cells such as epithelial cells, liver cells, fibroblasts, and kidney cells, nerve cells, immune cells, ES cells, primordial germ cells, sperm, and eggs, and include primary cultures of these cells and cell lines derived from these cells, i.e., cells proliferated from these cells.

[0069] Another aspect of the present invention is a method for screening candidate substances for the prevention and treatment of hypercholesterolemia and arteriosclerosis using the non-cloned miniature pig and cells of one aspect of the present invention. The screening method of one aspect of the present invention comprises the following steps: A step of administering a test substance to a non-cloned mini-pig according to one embodiment of the present invention or a cell derived from the non-cloned mini-pig as a test subject, and measuring the concentration of at least one type of cholesterol selected from the group consisting of LDL cholesterol and VLDL cholesterol or the degree of arteriosclerosis in the test subject; and A step of screening the test substance as a candidate substance for preventing or treating hypercholesterolemia or arteriosclerosis, using as an indicator whether the cholesterol concentration or the degree of arteriosclerosis is the same as or lower than that of a control.

[0070] In one embodiment of the screening method of the present invention, the control is preferably at least one type of control selected from the group consisting of the concentration and level before administration of the test substance, and the concentration and level obtained using a wild-type pig and cells derived from a wild-type pig instead of a non-cloned mini-pig and cells derived from the non-cloned mini-pig as test subjects.

[0071] The test substance is not particularly limited, but examples thereof include chemical substances, proteins, peptides, steroids, etc., which may be new or known, and may be produced by synthesis, extraction, or other methods, or may be commercially available products. The method of administering the test substance to the subject is not particularly limited, and examples thereof include oral administration, injection, infusion, and application when the subject is a pig; examples of methods include addition to a cell culture solution, administration into cells using a transfection reagent and electroporation.

[0072] The administration period, number of administrations and dosage of the test substance can be appropriately selected depending on the size, age, sex, symptoms, etc. of the test subject, and are not particularly limited. Conditions such as the rearing environment and age of the test subject pig and the culture method of the test subject cells may be appropriately set according to the desired test system. For example, the test subject pig may be fed a high-fat diet, but it is preferable to feed the pig a normal diet in order to suppress an excessive increase in the total cholesterol concentration.

[0073] The method for measuring the cholesterol concentration and the degree of arteriosclerosis in a subject may be carried out with reference to other items in this specification. For example, the concentration of LDL cholesterol or VLDL cholesterol in the plasma cholesterol section, and the presence or absence of arteriosclerosis may be measured. In addition to these measurements, the total cholesterol concentration in plasma and the concentration of HDL cholesterol in the cholesterol section, the total neutral fat concentration, and the concentrations of LDL, HDL, and VLDL in the neutral fat section may be measured, and other test items, such as blood glucose level, red blood cell volume, and white blood cell volume, may be measured and used as one of the judgment indicators for screening candidate substances.

[0074] The degree of arteriosclerosis may also be measured by ultrasound (echo), magnetic resonance imaging (MRI), or the like, or may be measured histochemically by taking a vascular tissue sample from a subject.

[0075] When the test subject is a cell, its proliferation rate, metabolic rate, intracellular LDL cholesterol and VLDL cholesterol concentrations, etc. can be measured by general biochemical and cell biological techniques.

[0076] In one embodiment of the screening method of the present invention, the preventive and therapeutic potential of a test substance against hypercholesterolemia and atherosclerosis is determined based on whether the cholesterol concentration and the degree of atherosclerosis are the same or lower than those of a control. That is, if the relatively high LDL cholesterol concentration and / or VLDL cholesterol concentration are maintained or reduced in non-cloned minipigs and cells, or if the atherosclerosis observed in non-cloned minipigs is maintained or improved, the candidate substance can be said to be useful as a preventive or therapeutic agent against hypercholesterolemia and atherosclerosis. When comparing with a control, the health condition of the test subject and the results of other test items may be taken into consideration.

[0077] In another embodiment of the present invention, there is a method of using organs, tissues and cells derived from the non-cloned miniature pig of one embodiment of the present invention for screening according to the purpose. Since these cells lack expression of the LDLR gene, they are useful in medical and biological research such as screening focusing on the LDLR gene and analysis of LDLR and related cell functions. For example, the cells of one embodiment of the present invention can be used to perform screening using the change in the signal factor to be analyzed as an index. Specifically, a method includes administering a test substance to the cells of one embodiment of the present invention as a test subject, examining the change in the signal factor in the test subject, and then screening a test substance that causes a change in the signal factor different from that of a control as a candidate substance. Factors involved in life phenomena involving LDLR and lipoprotein metabolism, and regulators of signal factors can be identified by screening.

[0078] The non-cloned minipigs of one embodiment of the present invention tend to fully reproduce the pathology of human hypercholesterolemia and atherosclerosis, and unlike normal pigs, are easy to handle even in long-term experiments. Therefore, they are expected to be widely used for screening of medicines for the diagnosis, treatment, and prevention of hypercholesterolemia and atherosclerosis, and for elucidating chronic side effects after treatment and their mechanisms of development.

[0079] Furthermore, based on the findings of the present inventors to date, the following can be said about the non-cloned miniature pig of one embodiment of the present invention. The SB lineage F3 homozygous (LDLR- / -) miniature pigs produced in the examples showed hyperlipidemia, even on a normal diet, similar to other hyperlipidemia model pigs. The total blood cholesterol concentration increased more than twice as much when fed a high-fat diet, and plaque was formed in the coronary artery after 4 months by intravascular ultrasound imaging. After coronary artery plaque was confirmed, various types of drug-eluting stents were placed in the plaque lesions, and the vascular reaction at the stent placement site was observed 3 months after placement. As a result, neointima was formed, and delayed vascular reactions such as infiltration of inflammatory cells in the neointima, fibrin accumulation around the stent struts, fragmented calcification, and foamy macrophages were observed in the neointima. Furthermore, the results were similar to those of changes after treatment with stent placement in human hypercholesterolemia and arteriosclerosis patients. Furthermore, because it is now possible to observe changes from 6 months to 2-3 years after stent treatment, which was previously difficult or impossible to observe in hyperlipidemic pigs bred from normal livestock, it has become possible to study the mechanisms of chronic thrombosis caused by new in-stent arteriosclerosis, which becomes a problem 1 year after placement, and new treatment methods for preventing these chronic complications, in second- and third-generation drug-eluting stents, disappearance changes in the stent structure made of bioabsorbable polylactide material, long-term biological reactions, and the mechanism of chronic thrombosis, which becomes a problem 1 year after placement, as well as new treatment methods for preventing these chronic complications. Furthermore, pathological analysis revealed arteriosclerotic plaque in the aorta and coronary artery, and scattered cholesterol crystals at the site, which has led to the development of the world's first large animal model of cholesterol thrombosis, making it possible to clarify the mechanism of its onset and treatment strategies.

[0080] In addition, in the SB lineage F3 generation homozygous (LDLR- / -) miniature pigs produced in the examples, signs of arteriosclerosis were observed not only in the aorta and coronary arteries but also in the fundus artery. A fundus camera was used to examine the correlation between fundus artery lesions and coronary artery lesions, and carotid artery intima thickening and proliferation of intramural nutrient vessels were observed. Therefore, a quantitative evaluation was made possible using contrast carotid artery ultrasound of the brightness change due to the progression of arteriosclerotic lesions accompanied by an increase in the common carotid artery intima-media complex thickness and the correlation with the histological proliferation of intramural nutrient vessels in the same area.

[0081] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be achieved. EXAMPLES

[0082] [1. Evaluation method] (1) Fertility survey The conception rate survey was carried out by visually confirming that signs of estrus did not reappear 21 days after artificial insemination (non-return method), and by confirming the amniotic sacs using an ultrasound imaging diagnostic device (Fujidaira Industrial Co., Ltd.: "HS-101V for pigs") 28 days after artificial insemination to determine the presence or absence of pregnancy. The conception rate was calculated as the proportion of sows that were confirmed to be pregnant among the artificially inseminated sows. The parturition rate was calculated as the proportion of sows that gave birth to at least one pig among the sows that were confirmed to be pregnant.

[0083] (2) Determining whether the offspring are homozygous or heterozygous The PCR method was used to determine the mutant allele and the wild-type allele by specifically detecting each of them. Specifically, genomic DNA was extracted from a pig ear using "NucleoSpin (registered trademark) Tissue" (manufactured by Takara Bio Inc.). PCR analysis was performed using "EmeraldAmp (registered trademark) PCR Master Mix" (manufactured by Takara Bio Inc.) with 40 ng of purified genomic DNA sample as a template in a 20 μl volume reaction solution. The PCR cycle was performed under the following conditions: first, 98 ° C for 3 minutes, then 98 ° C for 10 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute and 15 seconds, for a total of 35 cycles, and finally, 72 ° C for 10 minutes. FP2 and LDLRRex3F4 described in Patent Document 1 were used as primers for detecting the mutant allele. This primer set amplified a 1.1 kb mutant allele-specific product.

[0084] The detection of the wild-type allele was performed in the same manner as above, except that 100 ng of purified DNA was used as a template and LDLRRex3F4 and LDLRex4R1 described in Patent Document 1 were used as primers for detecting the wild-type allele.

[0085] Based on the combination of alleles detected using each primer set, homozygous and heterozygous types were determined.

[0086] (3) Blood tests Blood was collected from the porcine vein using a heparin blood collection tube, and hematological tests were performed. After the tests, the blood was centrifuged at 2,000 rpm for 15 minutes to separate and collect plasma. The collected plasma was used for blood biochemistry tests, and the four fractions of cholesterol and neutral fat were analyzed by the HPLC method described in Okazaki et al.'s paper (Arterioscler Thromb Vasc Biol 2005, 25: 578-584).

[0087] (4) Quality evaluation of frozen semen After freezing and thawing, the frozen semen was diluted again with improved Modena solution, and a drop of 100 μl of sperm suspension was prepared in a 3.5 cm petri dish and covered with liquid paraffin (light). 2 , 5% CO 2 The mixture was preincubated for 15 minutes in a gas-phase incubator at 38.5° C. After preincubation, the percentage of motile sperm in the petri dish was measured using a computerized sperm motility analyzer CASA system (manufactured by Air Brown; “CEROSII”).

[0088] (5) Intravascular ultrasound (IVUS) imaging and stent placement in coronary arteries After the pig was given general anesthesia, the sheath insertion site in the neck was shaved and disinfected, and under local anesthesia (10 ml of 1% lidocaine was administered subcutaneously), an arterial puncture was performed with the sheath to secure an arterial line. Under hemodynamic monitoring with an arterial pressure line and an electrocardiogram, the coronary artery was observed with a catheter inserted from the sheath and an intravascular image device. After confirming the characteristics and location of the plaque lesion in the coronary artery, the inner diameter of the lesioned area was measured with IVUS, and the size of the stent to be placed was determined. The stent was placed in the coronary artery with the outer diameter after stent expansion and the inner diameter of the lesioned area being 1.2:1. After the coronary artery catheter was operated, the device and sheath were removed, and after manually confirming compression and hemostasis, anesthesia was terminated.

[0089] (6) IVUS imaging after stent placement Coronary artery catheterization and intravascular imaging were performed on pigs under general anesthesia in the following manner. Coronary angiograms were taken using a Toshiba Medical Systems X-ray angiography system and recorded on a CD. IVUS images were taken by automatically withdrawing a Boston IVUS probe catheter using a mechanical pullback system at 0.5 mm per second and recording the images on a CD. Optical coherence tomography (OCT) images were taken using a St. Jude Medical catheter, which was withdrawn at a speed of 1 mm per second while flushing the vessel lumen with contrast agent, and the images were recorded on a hard disk. OCT and IVUS were used to qualitatively or quantitatively observe the thrombus and thrombus-intima changes in the stent.

[0090] (7) Histological analysis after stent placement After euthanasia, the pigs were perfused with physiological saline and fixed with 10% formalin, and tissues (heart, ascending and descending aorta, and carotid artery) were collected for histopathological examination. The hearts were excised, and the coronary arteries were dissected and embedded in resin or cut into paraffin sections for histopathological analysis.

[0091] [2. Creation of a high cholesterol / atherosclerosis model miniature pig] (1) Generation of homozygous S lineage (LDLR- / -) The applicant, National Agriculture and Food Research Organization, owns an F1 generation heterozygous LDLR-KO pig described in Patent Document 1 (LDLR+ / -; average body weight at 6 months of age, approximately 110 kg; average litter size, 10-12 pigs), and a miniature pig "Sakura Koyuki" (LDLR+ / +; average body weight at 9 months of age, approximately 40 kg; average litter size, approximately 5 pigs) purchased from the National Livestock Breeding Center were crossed by artificial insemination to produce an S line F1 generation.

[0092] Specifically, four LDLR-KO F1 generation heterozygous female pigs were introduced at about 6 months of age into an isolated pig house in the pig farming area of ​​the Saitama Agricultural Technology Research Center, and two male mini pigs were introduced into the isolated pig house in two separate batches. The isolated pig house meets the requirements for a genetic modification experimental facility. All of the introduced pigs were isolated for about one month, and blood was taken from the jugular vein immediately after introduction and one month after isolation, and antibodies to Aujeszky's disease and PRRS disease were tested for the pigs. Pigs that were confirmed to be antibody-negative and in good health were moved to a pig house that met the management standards for genetically modified animals.

[0093] The LDLR-KO pig F1 generation heterozygote was observed for estrus signs twice a day, in the morning and in the evening, to determine the day of estrus. Next, after confirming two to three estrus cycles, artificial insemination was performed using fresh semen collected from a miniature pig in time with the third to fourth estrus. S line F1 generation was produced by natural birth from the fertilized LDLR-KO pig F1 generation heterozygote. DNA was extracted from ear pieces collected at the time of ear numbering within one week after birth for the S line F1 generation, and the genotype of the LDLR gene (LDLR+ / -, LDLR+ / +) was confirmed by PCR. After confirming the genotype, heterozygote (LDLR+ / -) individuals (S line F1 generation heterozygote) were selected from the S line F1 generation.

[0094] The selected S line F1 generation heterotypes (LDLR+ / -) were crossed with each other to obtain the S line F2 generation. Next, heterotype individuals (S line F2 generation heterotypes) were selected from the S line F2 generation, and the S line F2 generation heterotypes (LDLR+ / -) were crossed with each other, or the S line F2 generation heterotypes (LDLR+ / -) and the S line F1 generation heterotypes (LDLR+ / -) to obtain the S line F3 generation, from which homotype S line F3 generation homotype (LDLR- / -) individuals were successfully produced. The obtained S line F3 generation homotype (LDLR- / -) was designated as the S line homotype (LDLR- / -).

[0095] However, as described below, the S line that was created had problems such as difficulty in producing and maintaining individuals, low litter size, high death rates immediately after birth, very low weaning rate on the 35th day after birth, and low disease resistance, leading to frequent sudden death or skin diseases in rearing pigs, regardless of the F1 to F3 generations. In addition, the S line had problems in that the piglets could not suckle due to enlarged nipples, making it impossible to continue a stable supply.

[0096] (2) Creation of a high cholesterol / atherosclerosis model miniature pig (SB strain homozygous (LDLR- / -)) The S strain homozygous (LDLR- / -) and a British Berkshire (LDLR+ / +; average weight at 8 months of age, approximately 100 kg; average litter size, approximately 8) were crossed by artificial insemination to produce the SB strain F1 generation.

[0097] Specifically, two homozygous (LDLR- / -) males from the S line F3 generation were used to artificially inseminate two female Berkshires, and the fertilized Berkshires were allowed to give birth naturally to produce the SB line F1 generation. DNA was extracted from ear pieces taken from the resulting SB line F1 generation when numbering the ears within one week of birth, and the genotype of the LDLR gene was confirmed by PCR. After confirming the genotype, relatively small heterozygous (LDLR+ / -) individuals (SB line F1 generation heterozygous) were selected from the SB line F1 generation.

[0098] The selected SB line F1 generation heterotypes (LDLR+ / -) were crossed with each other, or with the SB line F1 generation heterotypes (LDLR+ / -) and the miniature pigs (LDLR+ / +) used in 1 above, to obtain the SB line F2 generation. Next, the SB line F2 generation was divided into relatively small homozygous individuals (SB line F2 generation homozygous) and heterozygous individuals (SB line F2 generation heterozygous), and the SB line F3 generation was obtained by crossing the SB line F2 generation homozygous (LDLR- / -) with each other, or the SB line F2 generation homozygous (LDLR- / -) and the heterozygous (LDLR+ / -). Furthermore, the SB line F3 generation was selected for relatively small homozygous individuals (SB line F3 generation homozygous), and the SB line F4 generation, which was all homozygous, was obtained by crossing the SB line F3 generation homozygous (LDLR- / -) with each other. In the SB line F2 generation and SB line F4 generation, we succeeded in producing high cholesterol / atherosclerosis model miniature pigs that showed gene homogenization (LDLR- / -) and small body size.

[0099] (3) Specific production procedure (3-1) Semen collection and propagation using artificial insemination Because of the difference in size between the LDLR-KO pig F1 generation heterozygous (LDLR+ / -) described in Patent Document 1, which was produced from the large Landrace breed, and the miniature pig Sakura Koyuki (LDLR+ / +), so-called direct mating by natural mating was not possible. Therefore, semen was collected by the stealing method using medium-sized and small breeds in estrus or by the hand pressure method using a small dummy developed and made for miniature pigs, and an LDLR-KO pig F2 generation heterozygous (LDLR+ / -) was conceived and delivered by artificial insemination. In addition, when breeding the S line after the F1 generation, and when breeding the S line homozygous (LDLR- / -) with the British Berkshire (LDLR+ / +) to produce the SB line high cholesterol / arteriosclerosis model miniature pig, problems arose such as the boar's penis being difficult to insert into the vagina due to the narrowness of the vagina even when the female's body size became small, and semen not being able to enter the vagina, so artificial insemination was continuously used for breeding.

[0100] In the stealing method, a female pig in estrus was restrained by nasal restraint, and then 0.08 mg / kg of midazolam was injected intramuscularly as an analgesic and sedative based on body weight. After the boar mounted the female pig, a collector used manual pressure from the side to collect semen from the pig into a wide-mouth storage bottle with a volume of approximately 40 ml to 200 ml.

[0101] In the manual pressure method using a small dummy, the boar was placed on the small dummy, and then, as in the lateral collection method, the collector used manual pressure from the side to collect semen into a wide-mouth storage bottle in a volume of approximately 40ml to 200ml.

[0102] Immediately after collection, the semen was placed in a storage bottle in hot water at about 35°C, transported to the semen testing room, and diluted to the required amount with Modena solution. The diluted semen was stored in a semen storage room with temperature control at 15°C until just before artificial insemination. A portion of the semen was placed on a semen property testing board set on a heating plate heated to 38°C, observed at 200x using an optical microscope, and the motile sperm rate and sperm vitality were measured according to standard methods. For individuals confirmed to be in estrus, 50ml to 70ml of collected semen was loaded into a syringe twice, on the day of estrus and the day after, and artificial insemination was performed according to standard methods using an artificial insemination catheter (manufactured by Fujihira Kogyo Co., Ltd.) commonly used for pigs.

[0103] (3-2) Creation of a small dummy The small dummy was made by reducing all dimensions of a regular commercially available dummy. After creating the main body with a steel frame, the riding surface was created. The height of the steel frame was 60cm or less, the length was 70cm, and the width was 70cm or less. The riding surface was 12cm high, 60cm long, and 25cm wide. The riding surface was made from solid wood and was used as it was, or elastic sponge was wrapped around the frame and then covered with lining cloth.

[0104] (3-3) Feeding method From the first day after parturition until the next day, piglet milk replacer was melted in hot water for piglets who were unable to suckle, depending on the suckling condition of the newborn piglets. 5 ml of the melted piglet milk replacer was forcefully orally fed to each piglet using a 5 ml syringe. From the third day onwards, all piglets in the litter were fed the piglet milk replacer ad libitum in a feeding trough until two weeks after parturition.

[0105] (3-4) Feed The pigs in the maintenance group were fed normal feed (regular food) with the following fat content (based on the Japanese Standard Feed Composition Table), while the experimental group was fed a high-fat feed for four weeks after induction and then used in the experiment. 1) Maintenance group From the day after parturition to 2 weeks of age: Fat content 4.0% or more (Feed name: Nisshin Marubeni Pig Replacement Milk Sanikko No. 2) 3 weeks to 5 weeks old: Fat content 4.0% or more (Feed name: Northan Brand piglet formula feed early stage feed Weenie A) 5 weeks to 2 months: Fat content 3.5% or more (Feed name: Northern Brand piglet artificial milk late stage compound feed Winnie B) 2 months to 6 months: Fat content 2.5% or more (Feed name: Northern Meat Pig Feed for Raising Clear Meat Pig) 6 months or older: Fat content 2.0% or more (Feed name: Nextel Breed, a compound feed for raising Northern Indian pigs) 2) Experimental group From introduction to the end of the experiment: Fat content 15% beef tallow, 1.5% cholesterol (Feed name: FeedOne Co., Ltd. High fat special feed)

[0106] (3-5) Preparation of frozen semen Semen was collected from SB homozygous breeding boars by hand pressing using a small dummy, and frozen semen was prepared according to the method of Niwa et al. The collected semen, excluding the gelatinous pig, was immediately brought back to the laboratory in a warmed state at 38°C, and 2 / 3 of the collected amount of modified Modena solution was added. The semen was cooled from 38°C to 15°C in a semen low-temperature processing room for about 2 hours, then dispensed into 50 ml glass centrifuge tubes, centrifuged at 700G for 15 minutes while still cooled, the seminal plasma was removed with an aspirator, mixed with NSF-1 solution, and cooled to 5°C for 90 minutes. The cooled sperm suspension was added with the same amount of NSF-2 solution as NSF-1 solution, and the final glycerin concentration was adjusted to 3% and the final sperm concentration to 1 billion / ml, and then filled into 0.25 ml plastic straws using a single-type straw tube filling and sealing machine (Fujidaira Kogyo Co., Ltd.; "SFM-1"). The straws were precooled at 4 cm above the liquid nitrogen surface for approximately 20 minutes and then immersed in liquid nitrogen. All straws were stored in liquid nitrogen cylinders until use.

[0107] (3-6) Thawing frozen sperm and artificial insemination When estrus pigs were found in the morning, artificial insemination with frozen semen was performed twice, in the evening of the same day and in the morning of the following day. Four bottles of frozen semen were removed from the liquid nitrogen-filled cylinders and immediately immersed in 38°C warm water for about 10 seconds to thaw. The thawed semen was mixed with 1.25 ml of warmed improved Modena solution and filled into two 1 ml syringes to prepare a sperm suspension. For estrus pigs, after thoroughly washing the genitals, 1 ml of sperm suspension was injected into the deep uterine horn and uterine body using a catheter set for deep uterine injection (manufactured by Fujihira Kogyo Co., Ltd.; "Takumi"), and finally 3 ml of air was injected, completing one artificial insemination.

[0108] (3-7) General anesthesia method The pig was anesthetized and sedated by intramuscular injection of ketamine at 5 mg / kg. After intramuscular injection, the pig was secured in a supine position on a catheter stand after an intravenous route was established. The pig was intubated and given general anesthesia by inhaling 50% oxygen and 1%-4% sevoflurane while managing its breathing at a tidal volume of 10 ml / kg-15 ml / kg and 20 breaths / min.

[0109] [3. Reproductive performance of high cholesterol / atherosclerosis model miniature pigs] (1) Comparative evaluation of SB and S strains Over a seven-year period from FY2013 to FY2019, semen was collected from S lineage (F1 to F3 generations), SB lineage (F1 to F4 generations), and control male pigs by the intercept method or the manual pressure method using a small female pig dummy. 50 to 70 ml of each semen was artificially inseminated twice, on the day of estrus and the day after, into a total of 79 naturally estrus breeding female pigs, including 22 S lineage (F1 to F3 generations), 40 SB lineage (F1 to F4 generations), and 17 control pigs (Landrace), according to the origin of the semen. The number of conceptions and births of each type of female pig is summarized in Table 1.

[0110] [Table 1]

[0111] As shown in Table 1, the conception and farrowing rates of the S line were significantly lower than those of the SB line and the control.

[0112] As shown in Table 1, the number of pigs that gave birth to each type of sow was 16 for the S line (F1 to F3 generations), 36 for the SB line (F1 to F4 generations), and 15 for the control. The total number of piglets given birth to by these breeding sows was 117 for the S line (F1 to F3 generations), 350 for the SB line (F1 to F4 generations), and 130 for the control. From these results, the average number of piglets given birth to by one sow (average litter size) was calculated, and the piglets were weighed immediately after birth and on the 35th day after birth to obtain the average birth weight and average weaning weight. These results are shown in Table 2.

[0113] [Table 2]

[0114] As shown in Table 2, the SB line had a significantly higher average litter size than the S line. The SB line also had a comparable average birth weight to the S line, but was significantly lower than the control. The SB line also had a significantly lower average weaning weight than the S line and the control.

[0115] The height and diameter of all left and right nipples of 10 sows (5 from each generation of S line and 5 from SB line F3 generation) that had given birth twice or more were measured using a digital scale (Mitutoyo; "Digimatic Caliper"), and the average was calculated for each line. The results are shown in Table 3.

[0116] [Table 3]

[0117] As shown in Table 3, the average nipple height and average nipple diameter of the SB line were significantly smaller than those of the S line. This indicates that the nipples of the SB line are generally smaller and easier for piglets to suckle.

[0118] Taking the results of Tables 1 to 3 together, it was found that the SB line was characterized by high conception rate and parturition rate, large average litter size, small body weight, and easy suckling of piglets.

[0119] (2) Evaluation of the number and weight of offspring of each generation of SB line The average number of offspring born and the average body weight at 270 days of age for each generation of the SB line are shown in Tables 4 and 5.

[0120] [Table 4]

[0121] [Table 5]

[0122] As shown in Table 4, the average number of offspring from the SB line F2 generation onwards tended to be higher than those of the SB line F1 generation and the S line. The number of offspring in the SB line F4 generation was significantly higher than that of the S line F1 generation. In addition, as shown in Table 5, the average body weight at 270 days of the male and female pigs in the SB line F4 generation was similar to that of the S line F3 generation. Between each generation of both sexes, there was a tendency for the S line F1 generation to increase compared to the SB line F2 generation and the SB line F3 generation (P<0.06), but it decreased again in the SB line F4 generation. The average body weight at 270 days of the male pigs was smaller than that of the female pigs in each generation of the SB line.

[0123] (3) Viability assessment of each generation of SB lineage The stillbirth and immediate postnatal mortality rates of each generation of SB line piglets (F1 generation to F4 generation) are shown in Figure 1, and the preweaning mortality rates are shown in Figure 2, together with the S line (F1 generation to F3 generation).

[0124] As shown in Figure 1, the stillbirth and immediate postnatal mortality rates varied greatly between generations, and no significant differences were observed between groups. However, as shown in Figure 2, the preweaning mortality rate was clearly lower in the SB line than in the S line, and was significantly lower in the SB line F2 generation (mortality rate 20.0%), SB line F3 generation (mortality rate 10.2%), and SB line F4 generation (mortality rate 8.6%) than in the S line F1 generation (mortality rate 54.5%) (P<0.01).

[0125] From the above results, it was found that the mortality rate of the SB line decreased and the offspring grew well after weaning.

[0126] (4) Genotypic evaluation of each generation of SB lineage The genotypes of the SB lineage piglets (F1 to F4 generations) were determined to be wild type (LDLR+ / +), heterozygous type (LDLR+ / -), and homozygous type (LDLR- / -), and the results are shown in Figure 3 along with the results for the S lineage piglets (F1 to F3 generations).

[0127] As shown in FIG. 3, the wild type decreased and the homozygous type increased with each passage of the SB strain, and finally, in the F4 generation of the SB strain, we succeeded in homozygous genotype in all pigs.

[0128] In addition, the average kinship coefficient and average inbreeding coefficient of each generation of the SB line (F1 generation to F4 generation) were calculated together with the S line (F1 generation to F3 generation) using the calculation software "CoeFR ver.3.7" (Satoh, M., A program for computing inbreeding coefficients from large data sets. Journal of Swine Science). The results are shown in Table 6.

[0129] [Table 6]

[0130] As shown in Table 6, the average inbreeding coefficient decreased in the S line F3 generation due to the introduction of new minipigs, but increased in the SB line F3 generation due to gene homogenization. It decreased again in the SB line F4 generation due to the introduction of new British Berkshires. The average relatedness coefficient increased rapidly in the S line F2 generation and remained relatively stable in the SB line.

[0131] (5) Evaluation of semen quality in SB strains The quality and fertility of thawed frozen semen of the SB line were measured. The sperm motility before freezing and after freezing and thawing was evaluated using the CASA system. The results are shown in Table 7. In addition, the frozen-thawed homozygous (LDLR- / -) semen suspension of the SB line F3 generation was artificially inseminated into British Berkshire (LDLR+ / +) breeding female pigs using a deep uterine injection catheter (manufactured by Fujihira Kogyo Co., Ltd.; "Takumi Catheter"), and the results of conception rate, farrowing rate, and litter size are shown in Table 8.

[0132] [Table 7]

[0133] [Table 8]

[0134] As shown in Table 7, the motile sperm rate ("Motile Cells") in fresh semen before freezing was 79.5%, while the motile sperm rate after freezing and thawing was 19.1%. Also, as shown in Table 8, of the five artificially inseminated female pigs, four became pregnant, and three of them produced a total of 30 piglets. Also, as shown in Figure 4, the genotype of all the piglets born was LDLR+ / - heterozygous.

[0135] [4. Evaluation of high cholesterol / atherosclerosis miniature pig model] (1) Blood lipid assessment Feeding tests using normal or high-fat diets were conducted according to the following schedule. That is, the produced SB lineage F3 generation homozygous (LDLR- / -) miniature pigs were raised on normal feed until about 2 to 8 months of age. Then, the pigs were transported to the Nihon University School of Medicine experimental animal facility, and blood samples were taken from the vein for testing. Next, the pigs were fed a high-fat diet containing 1.5% cholesterol and 15% beef tallow at a rate of 1 kg per day for 4 months.

[0136] FIG. 5 shows the results of measuring the four fractions of blood cholesterol and neutral fat in the SB lineage SB lineage F3 generation homozygous (LDLR- / -) miniature pig, the homozygous (LDLR- / -) pig described in Patent Document 1, and the SB lineage. As shown in FIG. 5, the SB lineage F3 generation homozygous (LDLR- / -) miniature pig had the same blood count, biochemistry, and blood lipid profile under normal feeding as the homozygous (LDLR- / -) pig described in Patent Document 1. In addition, the high cholesterol / arteriosclerosis model miniature pig SB line created this time had high total cholesterol of 620 mg / dl and LDL cholesterol of 430 mg / dl, similar to the homozygous (LDLR- / -) pig described in Patent Document 1.

[0137] The results of measuring the transition of the average blood lipid concentration after feeding a high-fat diet for seven SB strain F3 generation homozygous (LDLR- / -) minipigs are shown in Figure 6. As shown in Figure 6, when the high-fat diet was fed for one month, the blood VLDL concentration increased by more than two-fold. The average blood count and biochemistry test results for nine SB strain F3 generation homozygous (LDLR- / -) minipigs before feeding a high-fat diet, 1 to 4 months after feeding a high-fat diet, and 12 months after stent placement are shown in Tables 9 and 10.

[0138] [Table 9]

[0139] [Table 10]

[0140] (2) Evaluation of intra-arterial atheroma formation SB strain F2 to F4 generation homozygous (LDLR- / -) miniature pigs were fed a high-fat diet for 4 months, and coronary artery atherogenesis was confirmed by intravascular imaging. The results are shown in Figure 7. No stenosis was observed in coronary angiography (Figure 7B) in Figure 7, but mild stenosis and plaque formation were observed in IVUS (Figure 7C to E).

[0141] The coronary artery and aorta were formalin fixed and HE stained for pathological diagnosis. The results are shown in Figure 8. As shown in Figure 8, plaque formation was confirmed by pathological diagnosis.

[0142] (3) Evaluation of intra-arterial atheroma formation after stent placement SB strain F2 to F4 generation homozygous (LDLR- / -) male miniature pigs were fed a high-fat diet for four months, and two types of stents were placed in the coronary arteries: a first-generation BVS (1stBVS, Abbott, "Absorb GT1 Bioabsorbable Scaffold System") and a second-generation stent (2ndDES, Abbott, "XIENCE Alpine").

[0143] Intravascular imaging and pathological evaluation were performed on pigs that were continuously fed a high-fat diet for 3 and 12 months after placement. The observation results after 12 months are shown in Figure 9. Coronary angiography in both the 1st BVS and 2nd DES sections after 3 months showed mild stenosis at the stent placement site. In addition, neointima was observed inside the stent when observed with IVUS.

[0144] Furthermore, in the pathological sections of the coronary arteries, infiltration of inflammatory cells in the neointima, fibrin accumulation around the stent struts, fragmented calcification, foamy macrophages, etc. were observed in both sections. As shown in Figure 9, in the observation after 12 months, mild stenosis was observed in coronary angiography in both the 1stBVS and 2ndDES sections, and new arteriosclerosis was observed in the stent in both sections in the pathological evaluation. A necrotic core layer and thrombus were observed in the 2ndDES section.

[0145] Three months after stent placement, the vascular response was investigated, and then the heart, ascending and descending aorta, and carotid artery were histopathologically examined after euthanasia. Neointima consisting mainly of smooth muscle cells was formed inside the stent, and inflammatory cell infiltration, fibrin accumulation around the stent struts, fragmented calcification, foamy macrophages, etc. were observed within the neointima, but no intra-stent thrombosis was observed.

[0146] (4) Evaluation of intracoronary atherosclerosis after stent placement A total of 12 animals, six each of homozygous (LDLR- / -) miniature pigs of the SB lineage F3 generation and LWD three-way crossbred pigs (Landrace x Large Yorkshire x Duroc), were fed a high-fat diet for four months, and a second-generation drug-eluting stent (2ndDES, Promus-PREMIER, Boston Scientific, Marlborough, MA) was placed in the coronary artery, and intravascular reactions were investigated histopathologically one month after placement. The results are shown in Figure 10.

[0147] As shown in Figure 10, in the LWD 3-way cross pigs, infiltration of many macrophages was observed around the stent struts, and no reaction similar to that seen in humans was observed. However, in the SB lineage F3 generation homozygous (LDLR- / -) miniature pigs, there was little inflammation and immature extracellular matrix in the neointima, similar to the early tissue reaction seen after stent placement in humans.

[0148] Male homozygous (LDLR- / -) minipigs of the SB strain F3 generation were fed a high-fat diet for 4 months. After coronary artery plaque was confirmed, a second-generation drug-eluting stent (2ndDES, Promus-PREMIER, Boston Scientific, Marlborough, MA) and a third-generation drug-eluting stent (3rdDES, SYNEGRY, Boston Scientific, Marlborough, MA) were placed in the coronary artery. One month after placement, the intravascular changes were compared with those of human specimens using OCT (Optical Coherence Tomography).

[0149] As a result, homogeneous neointima was observed after the placement of second- and third-generation drug-eluting stents in minipigs, and the thickness and area of ​​the neointima were significantly thinner in the third-generation stent, with the area being 1.90±0.03 mm. 2 , 2.05±0.07mm for the second generation 2 The area of ​​fibrin deposition around the stent struts was smaller in the third-generation drug-eluting stent in both minipigs and humans.

[0150] Twelve 3-month-old SB strain F3 homozygous (LDLR- / -) male miniature pigs were fed a high-fat diet for 4 months, and moderate stenosis of the coronary artery and eccentric fibrofatty plaque formation due to arteriosclerosis were confirmed by IVUS before stent placement. 2nd and 3rd DES were placed in the coronary artery of six pigs each, and OCT and tissue sections were prepared and evaluated 2 and 4 weeks after placement. The intravascular reaction results 4 weeks after placement of 2nd and 3rd generation DES are shown in Table 11.

[0151] [Table 11]

[0152] As shown in Table 11, the intravascular reaction after 4 weeks was stronger in the 2nd DES than in the 3rd DES, and the neointima thickness and fibrin deposition area were significantly lower in the 3rd DES than in the 2nd DES both 2 and 4 weeks after placement. A neointima with mature extracellular matrix and high density smooth muscle cells was observed in the 3rd DES.

[0153] In addition, SB lineage F2 to F4 homozygous (LDLR- / -) miniature pigs were fed a high-fat diet for 4 months, and the intravascular changes in the fundus artery and carotid artery were observed. A fundus camera was used to examine the correlation between fundus artery lesions and coronary artery lesions, and the retinal microglomeruli showed an amoeba-like shape. GFAP activity was also observed in the microglomeruli. A quantitative evaluation was performed using contrast carotid artery ultrasound to determine the correlation between the brightness change due to the progression of atherosclerotic lesions accompanied by an increase in the common carotid artery intima-media complex thickness and the histological proliferation of intramural nutrient vessels in the same area. Furthermore, proliferation of the adventitial carotid artery nutrient vessels (vv) appeared early in the development of atherosclerosis. [Industrial Applicability]

[0154] The non-cloned miniature pig of one embodiment of the present invention allows for stable production of robust pigs, and maintains the genotype of the LDLR gene homozygous (LDLR- / -) even after many generations, making it easy to maintain the lineage. In addition, the gene modification is performed without the use of a viral vector, and breeding and management can be performed in P3A facilities with double doors and escape prevention measures, so the hurdles in breeding are lower than those of genetically modified pigs developed so far, and breeding and breeding management can be performed by ordinary pig farmers and venture companies.

[0155] The non-cloned miniature pig of one embodiment of the present invention has high extrapolation properties, such as the ability to directly use treatment instruments and diagnostic devices developed for humans, and is highly versatile, as it is possible to screen short- to long-term biological changes and intravascular changes after treatment. It can be suitably used in a wide range of fields, such as the research field of hypercholesterolemia and arteriosclerosis, the field of disease model animals, elucidation of new diagnostic methods, therapeutic and preventive drugs, treatment methods and side effects after treatment, examination of materials for treatment instruments, and further development of retreatment methods.

Claims

1. The genotype of the LDLR gene is homozygous (- / -); A first miniature pig having a homozygous (- / -) genotype of the LDLR gene, which is obtained by artificially inseminating a nuclear transfer somatic cell clone pig derived from a Landrace pig and a Landrace x Large Yorkshire x Duroc three-way cross pig, and Sakura Koyuki having a wild (+ / +) genotype of the LDLR gene, and then subcultivating an SB line pig obtained by artificially inseminating a first miniature pig having a homozygous (- / -) genotype of the LDLR gene and a Berkshire pig having a wild (+ / +) genotype of the LDLR gene; and The average body weight at 270 days is 65 kg or less for males and 80 kg or less for females. Non-cloned mini pigs.

2. The non-cloned mini-pig of claim 1, wherein the non-cloned mini-pig has a conception rate of 80% or more, a farrowing rate of 80% or more, an average litter size of 8 or more, and / or an average pre-weaning mortality rate of 20% or less.

3. The non-cloned minipig according to any one of claims 1 to 2, wherein the non-cloned minipig has an average nipple height of 15 mm or less and / or an average nipple diameter of 12 mm or less.

4. The non-cloned miniature pig according to any one of claims 1 to 3, wherein the non-cloned miniature pig is used as a model animal for at least one disease selected from the group consisting of hypercholesterolemia and arteriosclerosis.

5. The non-cloned minipig of claim 4, wherein the hypercholesterolemia is type IIb hereditary hyperlipidemia.

6. The non-cloned minipig according to any one of claims 1 to 5, wherein the non-cloned minipig has a total cholesterol concentration in venous plasma of 220 mg / dl or more and / or an LDL cholesterol concentration in venous plasma of 140 mg / dl or more when fed a feed having a fat content of 2% to 5% by mass for two months.

7. a step of crossbreeding, by artificial insemination, a nuclear transfer somatic cell cloned pig having a heterozygous (+ / -) genotype of the LDLR gene and derived from a Landrace pig and a Landrace x Large Yorkshire x Duroc three-way crossbreed pig, with Sakura Koyuki having a wild (+ / +) genotype of the LDLR gene, and further subculturing the resulting S line pig to obtain a first miniature pig having a homozygous (- / -) genotype of the LDLR gene; a step of crossbreeding the first miniature pig with a Berkshire pig having a wild (+ / +) genotype of the LDLR gene by artificial insemination, and then subculturing the resulting SB line pig to obtain a non-cloned miniature pig having a homozygous (- / -) genotype of the LDLR gene; A method for producing a non-cloned miniature pig according to any one of claims 1 to 6, comprising:

8. A step of administering a test substance to a non-cloned miniature pig according to any one of claims 1 to 6 or a cell derived from the non-cloned miniature pig as a test subject, and measuring the concentration of at least one type of cholesterol selected from the group consisting of LDL cholesterol and VLDL cholesterol or the degree of arteriosclerosis in the test subject; screening the test substance as a candidate substance for preventing or treating hypercholesterolemia or arteriosclerosis based on whether the cholesterol concentration or the degree of arteriosclerosis is the same or lower than that of a control; A method for screening a candidate substance for the prevention or treatment of hypercholesterolemia or arteriosclerosis, comprising:

9. The screening method of claim 8, wherein the control is at least one type of control selected from the group consisting of the concentration and level before administration of the test substance, and the concentration and level when the test subject is a wild-type pig and cells derived from a wild-type pig.

10. An organ or cell derived from the non-cloned miniature pig according to any one of claims 1 to 6.

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