Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.
Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.
School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, 41566, Republic of Korea.
Hyo-Jin
HJ
Park
Center of Reproductive Medicine, Good Moonhwa Hospital, Busan, 48735, Republic of Korea.|Department of Biotechnology, Daegu University, Gyeongsan, 38453, Republic of Korea.
Sung-Hun
SH
Min
School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, 41566, Republic of Korea.|Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju, 55365, Korea.
Hoonsung
H
Choi
School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, 41566, Republic of Korea.
Junghyung
J
Park
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Sun-Uk
SU
Kim
Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju, 55365, Korea.
Seunghoon
S
Lee
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Sang-Rae
SR
Lee
Department of Animal Science, Division of Applied Life Science (BK21 Plus), Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Republic of Korea.
Il-Keun
IK
Kong
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Kyu-Tae
KT
Chang
Department of Biotechnology, Daegu University, Gyeongsan, 38453, Republic of Korea. dbkoo@daedu.ac.kr.
Deog-Bon
DB
Koo
School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, 41566, Republic of Korea. lee1@knu.ac.kr.
Dong-Seok
DS
Lee
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D004622:Embryo, Mammalian / Q000000981:diagnostic imaging; D047108:Embryonic Development; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000378:metabolism; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism; D053652:Nuclear Transfer Techniques
D008164:Luminescent Proteins; C413663:fluorescent protein 583
Embryonic development; Gene expression; Lentiviral infection; Mitochondria; Somatic cell nuclear transfer