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Vascular endothelium-specific overexpression of human catalase in cloned pigs

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Abstract

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H2O2), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H2O2 would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT–PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H2O2 in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H2O2 in vasodilation and in the mechanisms underlying vascular health and disease.

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Acknowledgments

This work is funded by a grant from the NIH (R24 RR018276-05) to MHL and RSP. The Tie2-hCat plasmid was kindly provided by Dr. George Kojda and Thao-Vi Dao of the Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität, Düsseldorf, Germany. Members of the R. S. Prather Lab were particularly helpful: Dr. Clay Isom, August Rieke, Dr. Jason Ross, Lee Spate, Dave Wax, and Dr. Jianguo Zhao. We are grateful for the assistance provided by members of the M. H. Laughlin Lab, including Jennifer Casati, David Harah, Dr. Rick McAllister, Ann Melloh, and Pam Thorne. We also thank Joyce Carafa of the MU Immunology and Cytology Core who assisted with FACS.

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Authors and Affiliations

  1. Department of Biomedical Sciences, University of Missouri, Columbia, MO, 65211, USA

    J. J. Whyte, J. Padilla, G. H. Simmons, A. A. Arce-Esquivel, S. B. Bender & M. H. Laughlin

  2. Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA

    M. Samuel, E. Mahan, K. M. Whitworth, Y. H. Hao, C. N. Murphy, E. M. Walters & R. S. Prather

  3. National Swine Resource and Research Center, University of Missouri, Columbia, MO, 65211, USA

    M. Samuel, E. M. Walters & R. S. Prather

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  1. J. J. Whyte
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  2. M. Samuel
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  3. E. Mahan
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  4. J. Padilla
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  13. M. H. Laughlin
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Correspondence to J. J. Whyte.

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Whyte, J.J., Samuel, M., Mahan, E. et al. Vascular endothelium-specific overexpression of human catalase in cloned pigs. Transgenic Res 20, 989–1001 (2011). https://doi.org/10.1007/s11248-010-9473-7

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