Gene silencing in bovine zygotes: siRNA transfection versus microinjection
Ciara M. O’Meara A , James D. Murray B , Solomon Mamo A , Emma Gallagher A , James Roche A and Patrick Lonergan A C
+ Author Affiliations
- Author Affiliations
A School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
B Departments of Animal Science and Population Health and Reproduction, University of California, Davis, CA 95616, USA.
C Corresponding author. Email: pat.lonergan@ucd.ie
Reproduction, Fertility and Development 23(4) 534-543 https://doi.org/10.1071/RD10175
Submitted: 12 July 2010 Accepted: 10 November 2010 Published: 11 April 2011
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 1 h 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).
Additional keywords: embryos, IVF, RNA interference.
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