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Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

Ozawa M, Himaki T, Ookutsu S, Mizobe Y, Ogawa J, Miyoshi K, Yabuki A, Fan J, Yoshida M - PLoS ONE (2015)

Bottom Line: However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models.Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine.More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT
High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

No MeSH data available.


Related in: MedlinePlus

Immunoblot analysis of transgenic pig plasma apo(a).Aliquots of plasma were separated by either 4% nondenaturing polyacrylamide gel electrophoresis (PAGE) or 4% SDS-PAGE under nonreducing (left) or reducing (right) conditions. The SDS-PAGE samples of nonreducing and reducing conditions were electrophoresed on the same gels. After electrophoretic transfer, the proteins were immunoblotted using an anti-human apo(a) mAb or an anti-apoB mAb as described in Materials and Methods. Since the reactivity of materials separated under reducings to anti-apo(a) mAb become weak, they were blotted with anti-HA mAb.
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pone.0132155.g004: Immunoblot analysis of transgenic pig plasma apo(a).Aliquots of plasma were separated by either 4% nondenaturing polyacrylamide gel electrophoresis (PAGE) or 4% SDS-PAGE under nonreducing (left) or reducing (right) conditions. The SDS-PAGE samples of nonreducing and reducing conditions were electrophoresed on the same gels. After electrophoretic transfer, the proteins were immunoblotted using an anti-human apo(a) mAb or an anti-apoB mAb as described in Materials and Methods. Since the reactivity of materials separated under reducings to anti-apo(a) mAb become weak, they were blotted with anti-HA mAb.

Mentions: In the human Lp(a) particle, apo(a) is covalently linked to apoB-100 by the presence of a single disulfide bridge [33]. It has been demonstrated that apo(a) and apoB-100 are present in a 1:1 molar ratio in Lp(a) [34]. To determine whether the apo(a) in the pig plasma was incorporated into a lipoprotein particle, total lipoproteins were subjected to electrophoresis on a 4% nondenaturing polyacrylamide gel (Fig 4, left panels). The apo(a) immunoreactive material in pig plasma migrated to a position that was identical to the position of apoB recognized by anti-apoB antibody. The HA tag was added at the C-terminus of apo(a) and the HA immunoreactive material in pig plasma also migrated to the position of apoB. Co-migration of apo(a) and apoB strongly suggested that they were in the same complex. To determine whether apo(a) was disulfide linked to pig apoB, the pig plasma were subjected to electrophoresis on a 4% denaturing polyacrylamide gel (SDS-PAGE) after boiling in the presence 2.3% SDS alone (nonreducing conditions) or in the presence of 2.3% SDS and 5% 2-mercaptoethanol (reducing conditions) (Fig 4, right panels). When the plasma samples were analyzed under nonreducing conditions, apo(a) migrated to a position that was identical to the position of apoB. Since after reduction with 2-mercaptoethanol, the reactivity of the materials to anti-apo(a) antibody was significantly reduced, we used anti-HA antibody to detect apo(a) under reducing condition. The epitope may be dependent on disulfide bonds. Under reducing conditions, two closely migrating immunoreactive bands and a single slightly faster migrating band were detected with anti-HA and anti-apoB antibodies, respectively. These bands migrated much faster than the bands detected under nonreducing conditions. These results suggest that the apo(a) in the transgenic pig circulates as a complex disulfide linked to apoB in plasma.


Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

Ozawa M, Himaki T, Ookutsu S, Mizobe Y, Ogawa J, Miyoshi K, Yabuki A, Fan J, Yoshida M - PLoS ONE (2015)

Immunoblot analysis of transgenic pig plasma apo(a).Aliquots of plasma were separated by either 4% nondenaturing polyacrylamide gel electrophoresis (PAGE) or 4% SDS-PAGE under nonreducing (left) or reducing (right) conditions. The SDS-PAGE samples of nonreducing and reducing conditions were electrophoresed on the same gels. After electrophoretic transfer, the proteins were immunoblotted using an anti-human apo(a) mAb or an anti-apoB mAb as described in Materials and Methods. Since the reactivity of materials separated under reducings to anti-apo(a) mAb become weak, they were blotted with anti-HA mAb.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492603&req=5

pone.0132155.g004: Immunoblot analysis of transgenic pig plasma apo(a).Aliquots of plasma were separated by either 4% nondenaturing polyacrylamide gel electrophoresis (PAGE) or 4% SDS-PAGE under nonreducing (left) or reducing (right) conditions. The SDS-PAGE samples of nonreducing and reducing conditions were electrophoresed on the same gels. After electrophoretic transfer, the proteins were immunoblotted using an anti-human apo(a) mAb or an anti-apoB mAb as described in Materials and Methods. Since the reactivity of materials separated under reducings to anti-apo(a) mAb become weak, they were blotted with anti-HA mAb.
Mentions: In the human Lp(a) particle, apo(a) is covalently linked to apoB-100 by the presence of a single disulfide bridge [33]. It has been demonstrated that apo(a) and apoB-100 are present in a 1:1 molar ratio in Lp(a) [34]. To determine whether the apo(a) in the pig plasma was incorporated into a lipoprotein particle, total lipoproteins were subjected to electrophoresis on a 4% nondenaturing polyacrylamide gel (Fig 4, left panels). The apo(a) immunoreactive material in pig plasma migrated to a position that was identical to the position of apoB recognized by anti-apoB antibody. The HA tag was added at the C-terminus of apo(a) and the HA immunoreactive material in pig plasma also migrated to the position of apoB. Co-migration of apo(a) and apoB strongly suggested that they were in the same complex. To determine whether apo(a) was disulfide linked to pig apoB, the pig plasma were subjected to electrophoresis on a 4% denaturing polyacrylamide gel (SDS-PAGE) after boiling in the presence 2.3% SDS alone (nonreducing conditions) or in the presence of 2.3% SDS and 5% 2-mercaptoethanol (reducing conditions) (Fig 4, right panels). When the plasma samples were analyzed under nonreducing conditions, apo(a) migrated to a position that was identical to the position of apoB. Since after reduction with 2-mercaptoethanol, the reactivity of the materials to anti-apo(a) antibody was significantly reduced, we used anti-HA antibody to detect apo(a) under reducing condition. The epitope may be dependent on disulfide bonds. Under reducing conditions, two closely migrating immunoreactive bands and a single slightly faster migrating band were detected with anti-HA and anti-apoB antibodies, respectively. These bands migrated much faster than the bands detected under nonreducing conditions. These results suggest that the apo(a) in the transgenic pig circulates as a complex disulfide linked to apoB in plasma.

Bottom Line: However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models.Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine.More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT
High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

No MeSH data available.


Related in: MedlinePlus