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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

The transgenic miniature pig and his offspring.A, pedigree of the transgenic miniature pig clone expressing human apo(a). Two offspring (No. 1 and 6) were dead when they were found, and could not be analyzed. B, Identification of the transgene, apo(a), in genomic DNA of offspring by PCR. The arrow indicates the position of the PCR product. C, Photograph of apo(a)-positive offspring (No. 2 and 5).
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pone.0132155.g003: The transgenic miniature pig and his offspring.A, pedigree of the transgenic miniature pig clone expressing human apo(a). Two offspring (No. 1 and 6) were dead when they were found, and could not be analyzed. B, Identification of the transgene, apo(a), in genomic DNA of offspring by PCR. The arrow indicates the position of the PCR product. C, Photograph of apo(a)-positive offspring (No. 2 and 5).

Mentions: In the second series of experiments, we finally succeeded in obtaining a piglet that is PCR-positive for human apo(a) and exhibited a high level of Lp(a) in plasma (Table 2). This piglet was used as a founder to establish a apo(a)-transgenic line of miniature pigs (Fig 3A). Six piglets were produced from a sow after artificial insemination using semen from the PCR-positive boar (Minidora), of which two died for unknown reasons, but seemed to be killed by their mother by crashing, and could not be analyzed. Two of the four living piglets were positive in PCR analysis, whereas the other two were negative (Fig 3A, 3B, and 3C). The levels of Lp(a) in the PCR-positive founder and two PCR-positive offspring on a standard chow diet were 682 mg/dL, 427 mg/dL, and 514 mg/dL, respectively (Table 2). These values are much higher than the levels of Lp(a) in three nontransgenic female pigs and the two PCR-negative offspring. Expression of human apo(a) in transgenic pigs did not result in a significant change in plasma total cholesterol, triglycerides, LDL-C levels, or HDL-C levels (Table 2).


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)

The transgenic miniature pig and his offspring.A, pedigree of the transgenic miniature pig clone expressing human apo(a). Two offspring (No. 1 and 6) were dead when they were found, and could not be analyzed. B, Identification of the transgene, apo(a), in genomic DNA of offspring by PCR. The arrow indicates the position of the PCR product. C, Photograph of apo(a)-positive offspring (No. 2 and 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132155.g003: The transgenic miniature pig and his offspring.A, pedigree of the transgenic miniature pig clone expressing human apo(a). Two offspring (No. 1 and 6) were dead when they were found, and could not be analyzed. B, Identification of the transgene, apo(a), in genomic DNA of offspring by PCR. The arrow indicates the position of the PCR product. C, Photograph of apo(a)-positive offspring (No. 2 and 5).
Mentions: In the second series of experiments, we finally succeeded in obtaining a piglet that is PCR-positive for human apo(a) and exhibited a high level of Lp(a) in plasma (Table 2). This piglet was used as a founder to establish a apo(a)-transgenic line of miniature pigs (Fig 3A). Six piglets were produced from a sow after artificial insemination using semen from the PCR-positive boar (Minidora), of which two died for unknown reasons, but seemed to be killed by their mother by crashing, and could not be analyzed. Two of the four living piglets were positive in PCR analysis, whereas the other two were negative (Fig 3A, 3B, and 3C). The levels of Lp(a) in the PCR-positive founder and two PCR-positive offspring on a standard chow diet were 682 mg/dL, 427 mg/dL, and 514 mg/dL, respectively (Table 2). These values are much higher than the levels of Lp(a) in three nontransgenic female pigs and the two PCR-negative offspring. Expression of human apo(a) in transgenic pigs did not result in a significant change in plasma total cholesterol, triglycerides, LDL-C levels, or HDL-C levels (Table 2).

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