title: Gene silencing in bovine zygotes: siRNA transfection versus microinjection.
abstract: The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P<0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P<0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P<0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P<0.05).
authors.affiliation: School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland.
authors.forename: Ciara M
authors.initials: CM
authors.lastname: O'Meara
authors.forename: James D
authors.initials: JD
authors.lastname: Murray
authors.forename: Solomon
authors.initials: S
authors.lastname: Mamo
authors.forename: Emma
authors.initials: E
authors.lastname: Gallagher
authors.forename: James
authors.initials: J
authors.lastname: Roche
authors.forename: Patrick
authors.initials: P
authors.lastname: Lonergan
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015820:Cadherins / Q000037:antagonists & inhibitors / Q000235:genetics; D002417:Cattle / Q000196:embryology / Q000235:genetics; D002478:Cells, Cultured; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D005260:Female; D005307:Fertilization in Vitro; D020868:Gene Silencing / Q000502:physiology*; D018014:Gene Transfer Techniques; D008297:Male; D008845:Microinjections / Q000379:methods*; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D014162:Transfection / Q000379:methods*; D015053:Zygote / Q000166:cytology / Q000378:metabolism*
chemical_list: D015820:Cadherins; D034741:RNA, Small Interfering