Production of cloned pigs with targeted attenuation of gene expression
| Autor: | Marcelo S. Albornoz, Vilceu Bordignon, Denyse Laurin, Bernardo Garziera Gasperin, Luis B. Agellon, Mario A. Martinez-Diaz, Carine Schneider, Nayla El-Beirouthi, David Zadworny |
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| Rok vydání: | 2012 |
| Předmět: |
Male
Nuclear Transfer Techniques Embryology Anatomy and Physiology Swine Agricultural Biotechnology lcsh:Medicine Small hairpin RNA 0302 clinical medicine RNA interference Pregnancy Reproductive Physiology Gene expression Gene Knockdown Techniques Biological Systems Engineering lcsh:Science Regulation of gene expression 0303 health sciences Multidisciplinary Expression vector Genetically Modified Organisms Stem Cells Agriculture Cell Differentiation Female RNA Interference Research Article Biotechnology Cell Physiology Cloning Organism Cell Potency Animal Types Biomedical Engineering Bioengineering Biology Large Animals Transfection 03 medical and health sciences Apolipoproteins E Animals Gene 030304 developmental biology Messenger RNA Granulosa Cells Base Sequence lcsh:R Reproductive System Fibroblasts Molecular biology Gene Expression Regulation lcsh:Q Veterinary Science 030217 neurology & neurosurgery Developmental Biology |
| Zdroj: | PLoS ONE PLoS ONE, Vol 8, Iss 5, p e64613 (2013) |
| ISSN: | 1932-6203 |
| Popis: | The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45–82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species. |
| Databáze: | OpenAIRE |
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