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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

Expression of human apo(a) in SCNT embryos and tissues of transgenic piglets.A, SCNT embryos after 7 d culture were stained with anti-HA antibody (left) or Hoechst 33342 (right). Bars, 50 μm. B, Paraffin sections were stained with anti-HA antibody, followed by HRP-conjugated secondary antibody and DAB substrate. Finally, the sections were stained with hematoxylin and mounted. The left and right panels show tissue from transgenic and non-transgenic piglets, respectively. a) brain, b) lung, c) heart, d) liver, e) kidney, f) stomach, g) small intestine, h) large intestine, i) testis. Bars, 100 μm. C, RT-PCR analysis showing expression of human apo(a) mRNA in the kidney of three transgenic piglets. Lanes: M, 100 bp DNA ladder markers; 1, kidney of transgenic piglet #1; 2, kidney of transgenic piglet #2; 3, kidney of transgenic piglet #3; 4, pig kidney epithelial cells stably expressing human apo(a) used as donor of SCNT; 5, kidney of non-transgenic pig; 6, liver of non-transgenic piglet. A band of 233 bp, corresponding to the β-actin (arrowhead) was detected in all samples, whereas a band of 158 bp, corresponding to the human apo(a) transcripts (arrow), is found in transgenic samples but not in non-transgenic samples. D, RT-PCR analysis showing expression of human apo(a) mRNA in the organs of a transgenic piglet. Bands of 233 bp (β-actin, arrowhead) and 158 bp (human apo(a), arrow) are visible. Lanes: M, 100 bp DNA ladder markers; 1, brain; 2, lung; 3, heart; 4, aorta; 5, muscle; 6, liver; 7, kidney; 8, pancreas; 9, spleen; 10, stomach; 11, small intestine; 12, large intestine; 13, skin.
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pone.0132155.g002: Expression of human apo(a) in SCNT embryos and tissues of transgenic piglets.A, SCNT embryos after 7 d culture were stained with anti-HA antibody (left) or Hoechst 33342 (right). Bars, 50 μm. B, Paraffin sections were stained with anti-HA antibody, followed by HRP-conjugated secondary antibody and DAB substrate. Finally, the sections were stained with hematoxylin and mounted. The left and right panels show tissue from transgenic and non-transgenic piglets, respectively. a) brain, b) lung, c) heart, d) liver, e) kidney, f) stomach, g) small intestine, h) large intestine, i) testis. Bars, 100 μm. C, RT-PCR analysis showing expression of human apo(a) mRNA in the kidney of three transgenic piglets. Lanes: M, 100 bp DNA ladder markers; 1, kidney of transgenic piglet #1; 2, kidney of transgenic piglet #2; 3, kidney of transgenic piglet #3; 4, pig kidney epithelial cells stably expressing human apo(a) used as donor of SCNT; 5, kidney of non-transgenic pig; 6, liver of non-transgenic piglet. A band of 233 bp, corresponding to the β-actin (arrowhead) was detected in all samples, whereas a band of 158 bp, corresponding to the human apo(a) transcripts (arrow), is found in transgenic samples but not in non-transgenic samples. D, RT-PCR analysis showing expression of human apo(a) mRNA in the organs of a transgenic piglet. Bands of 233 bp (β-actin, arrowhead) and 158 bp (human apo(a), arrow) are visible. Lanes: M, 100 bp DNA ladder markers; 1, brain; 2, lung; 3, heart; 4, aorta; 5, muscle; 6, liver; 7, kidney; 8, pancreas; 9, spleen; 10, stomach; 11, small intestine; 12, large intestine; 13, skin.

Mentions: Experiments were repeated four times. The fusion rate of SCNT embryos reconstituted with transgenic kidney epithelial cells was 77.8% (n = 200). The rate of cleavage, blastocyst formation rate, and mean blastocyst cell number were 80.2 ± 4.3%, 18.8 ± 3.9%, and 39.7 ± 9.4, respectively. Furthermore, all blastocysts (n = 38) expressed human apo(a) as assessed by immunohistochemistry (Fig 2A). Thus, the selected donor cells do not undergo the epigenetic reprogramming that would take place under early embryonic conditions.


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)

Expression of human apo(a) in SCNT embryos and tissues of transgenic piglets.A, SCNT embryos after 7 d culture were stained with anti-HA antibody (left) or Hoechst 33342 (right). Bars, 50 μm. B, Paraffin sections were stained with anti-HA antibody, followed by HRP-conjugated secondary antibody and DAB substrate. Finally, the sections were stained with hematoxylin and mounted. The left and right panels show tissue from transgenic and non-transgenic piglets, respectively. a) brain, b) lung, c) heart, d) liver, e) kidney, f) stomach, g) small intestine, h) large intestine, i) testis. Bars, 100 μm. C, RT-PCR analysis showing expression of human apo(a) mRNA in the kidney of three transgenic piglets. Lanes: M, 100 bp DNA ladder markers; 1, kidney of transgenic piglet #1; 2, kidney of transgenic piglet #2; 3, kidney of transgenic piglet #3; 4, pig kidney epithelial cells stably expressing human apo(a) used as donor of SCNT; 5, kidney of non-transgenic pig; 6, liver of non-transgenic piglet. A band of 233 bp, corresponding to the β-actin (arrowhead) was detected in all samples, whereas a band of 158 bp, corresponding to the human apo(a) transcripts (arrow), is found in transgenic samples but not in non-transgenic samples. D, RT-PCR analysis showing expression of human apo(a) mRNA in the organs of a transgenic piglet. Bands of 233 bp (β-actin, arrowhead) and 158 bp (human apo(a), arrow) are visible. Lanes: M, 100 bp DNA ladder markers; 1, brain; 2, lung; 3, heart; 4, aorta; 5, muscle; 6, liver; 7, kidney; 8, pancreas; 9, spleen; 10, stomach; 11, small intestine; 12, large intestine; 13, skin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132155.g002: Expression of human apo(a) in SCNT embryos and tissues of transgenic piglets.A, SCNT embryos after 7 d culture were stained with anti-HA antibody (left) or Hoechst 33342 (right). Bars, 50 μm. B, Paraffin sections were stained with anti-HA antibody, followed by HRP-conjugated secondary antibody and DAB substrate. Finally, the sections were stained with hematoxylin and mounted. The left and right panels show tissue from transgenic and non-transgenic piglets, respectively. a) brain, b) lung, c) heart, d) liver, e) kidney, f) stomach, g) small intestine, h) large intestine, i) testis. Bars, 100 μm. C, RT-PCR analysis showing expression of human apo(a) mRNA in the kidney of three transgenic piglets. Lanes: M, 100 bp DNA ladder markers; 1, kidney of transgenic piglet #1; 2, kidney of transgenic piglet #2; 3, kidney of transgenic piglet #3; 4, pig kidney epithelial cells stably expressing human apo(a) used as donor of SCNT; 5, kidney of non-transgenic pig; 6, liver of non-transgenic piglet. A band of 233 bp, corresponding to the β-actin (arrowhead) was detected in all samples, whereas a band of 158 bp, corresponding to the human apo(a) transcripts (arrow), is found in transgenic samples but not in non-transgenic samples. D, RT-PCR analysis showing expression of human apo(a) mRNA in the organs of a transgenic piglet. Bands of 233 bp (β-actin, arrowhead) and 158 bp (human apo(a), arrow) are visible. Lanes: M, 100 bp DNA ladder markers; 1, brain; 2, lung; 3, heart; 4, aorta; 5, muscle; 6, liver; 7, kidney; 8, pancreas; 9, spleen; 10, stomach; 11, small intestine; 12, large intestine; 13, skin.
Mentions: Experiments were repeated four times. The fusion rate of SCNT embryos reconstituted with transgenic kidney epithelial cells was 77.8% (n = 200). The rate of cleavage, blastocyst formation rate, and mean blastocyst cell number were 80.2 ± 4.3%, 18.8 ± 3.9%, and 39.7 ± 9.4, respectively. Furthermore, all blastocysts (n = 38) expressed human apo(a) as assessed by immunohistochemistry (Fig 2A). Thus, the selected donor cells do not undergo the epigenetic reprogramming that would take place under early embryonic conditions.

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