Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development = 형질전환 체세포를 이용한 복제수정란에서의 초기 배 발생 시기의 미토콘드리아 DNA 확인

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Title
Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development = 형질전환 체세포를 이용한 복제수정란에서의 초기 배 발생 시기의 미토콘드리아 DNA 확인
Author(s)
H J Park; S H Min; H Choi; J Park; Sun-Uk Kim; S Lee; Sang-Rae Lee; I K Kong; Kyu Tae Chang; D B Koo; D S Lee
Bibliographic Citation
in Vitro Cellular & Developmental Biology-Animal, vol. 52, no. 8, pp. 812-822
Publication Year
2016
Abstract
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.
Keyword
Embryonic developmentGene expressionLentiviral infectionMitochondriaSomatic cell nuclear transfer
ISSN
1071-2690
Publisher
Springer
DOI
http://dx.doi.org/10.1007/s11626-016-0053-x
Type
Article
Appears in Collections:
Ochang Branch Institute > Division of Bioinfrastructure > Futuristic Animal Resource & Research Center > 1. Journal Articles
Ochang Branch Institute > Division of Bioinfrastructure > National Primate Research Center > 1. Journal Articles
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