title: Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.
abstract: Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.
authors.affiliation: Heilongjiang Acamedy of Agricultural Sciences Postdoctoral Programme, Northeast Forestry University Postdoctoral Programme, Harbin, China.|Institute of Animal Husbandry Research, HeiLongJiang Academy of Agricultural Sciences, Harbin, 150086, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Fu
authors.affiliation: College of Animal Science, Northeast Agriculture University, Harbin, 150030, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Ren
authors.affiliation: Institute of Animal Husbandry Research, HeiLongJiang Academy of Agricultural Sciences, Harbin, 150086, China.|College of Animal Science, Northeast Agriculture University, Harbin, 150030, China.
authors.forename: Di
authors.initials: D
authors.lastname: Liu
authors.affiliation: Heilongjiang Acamedy of Agricultural Sciences Postdoctoral Programme, Northeast Forestry University Postdoctoral Programme, Harbin, China.
authors.forename: Jian-Zhang
authors.initials: JZ
authors.lastname: Ma
authors.affiliation: Heilongjiang Acamedy of Agricultural Sciences Postdoctoral Programme, Northeast Forestry University Postdoctoral Programme, Harbin, China.
authors.forename: Tie-Zhu
authors.initials: TZ
authors.lastname: An
authors.affiliation: College of Animal Science, Northeast Agriculture University, Harbin, 150030, China.
authors.forename: Xiu-Qin
authors.initials: XQ
authors.lastname: Yang
authors.affiliation: Institute of Animal Husbandry Research, HeiLongJiang Academy of Agricultural Sciences, Harbin, 150086, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Ma
authors.affiliation: Institute of Animal Husbandry Research, HeiLongJiang Academy of Agricultural Sciences, Harbin, 150086, China.
authors.forename: Zhen-Hua
authors.initials: ZH
authors.lastname: Guo
authors.affiliation: College of Animal Science, Northeast Agriculture University, Harbin, 150030, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Modern Education Technology and Information center, Heilongjiang University of Chinese Medicine, Harbin, 150040, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002232:Carbocyanines / Q000737:chemistry; D019976:Cloning, Organism; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D020868:Gene Silencing; D005818:Genetic Engineering; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D053768:Metal Nanoparticles; D053652:Nuclear Transfer Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000032:analysis* / Q000235:genetics; D013552:Swine; D014019:Tissue Donors; D019076:Transgenes
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D012337:RNA, Ribosomal, 18S; C089187:cyanine dye 3
keywords: RNA detection; development; nanoparticle; somatic cell nuclear transfer