131 GENE SILENCING IN BOVINE ZYGOTES: SIRNA TRANSFECTION v. MICROINJECTION

Abstract

Ribonucleic acid interference (RNAi) has become an effective tool for studying gene function in a variety of cells. The objective of this study was to compare the efficiency of gene silencing when siRNA were introduced into bovine zygotes by microinjection (as done previously; Tesfaye D et al. 2007 Mol. Reprod. Dev. 74, 978-988) v. a novel method of transfection in terms of gene knockdown and embryo development. For microin-jection, in vitro-produced bovine zygotes (16 h post insemination) were randomly assigned to 1 of 3 groups over 2 experiments. In Experiment 1, E-cadherin siRNA was injected at 100 μM (n = 168) and compared with PBS-injected (n = 180) and noninjected controls (n = 152). In Experiment 2, E-cadherin siRNA was injected at 375 μM (n = 154) and compared with PBS-injected (n = 136) and noninjected controls (n = 151). Embryos were subsequently cultured in vitro until Day 7 (day of IVF = Day 0). For transfection, the zona pellucida was removed from in vitro-produced zygotes. Zona-free zygotes were randomly assigned to 1 of 4 groups (i) GAPDH (n = 67), (ii) scrambled (n = 66), (iii) E-cadherin (n = 69) siRNA treatments at 100 nM or (iv) nontransfected controls (n = 66). Zygotes were incubated in transfection medium with siRNA for 1 h at 39°C, cultured individually in the well-of-the-well system to Day 7. The proportion of zygotes undergoing cleavage and developing to the blastocyst stage was recorded, and Day 7 embryos were frozen individually for mRNA analysis. Data for mRNA expression were fitted to a general linear model, and developmental stages were tested using ANOVA. Microinjection of 100 μM E-cadherin siRNA had no effect on phenotype (P > 0.05). 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%, 45.5%, and 62.9%, respectively) and blastocyst stage (39.0%, 32.5%, and 45%, respectively) compared with noninjected controls (P < 0.05). The mRNA abundance of the target gene was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 μM and 375 μM compared with control and PBS-injected groups (P < 0.05). Transfection with E-cadherin siRNA decreased development of 8-cell embryos (20.3 v. 53.0%, respectively) and blastocysts (7.2 v. 18.2%, respectively) compared with controls (P < 0.05). The mRNA relative abundance was not different between controls (nontransfected, or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100nM E-cadherin siRNA led to a 70% reduction in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05). Zona removal and transfection resulted in decreased embryo development compared with microinjection (P < 0.05). However, transfection yielded more efficient gene silencing of E-cadherin mRNA with reduced embryo development compared with microinjection. This technique of gene silencing could improve the efficiency of gene function studies in early bovine embryogenesis.

Supported by Science Foundation Ireland.