Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos.
The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.
Miyoshi
Kazuchika
K
Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan. kmiyoshi@agri.kagoshima-u.ac.jp
Mori
Hironori
H
Mizobe
Yamato
Y
Akasaka
Eri
E
Ozawa
Akio
A
Yoshida
Mitsutoshi
M
Sato
Masahiro
M
D000822:Animal Husbandry / Q000379:methods; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D047108:Embryonic Development; D005260:Female; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013552:Swine; D013556:Swine, Miniature / Q000196:embryology*; D014162:Transfection / Q000662:veterinary
D050814:Octamer Transcription Factor-3; D049452:Green Fluorescent Proteins