334 EFFECT OF CYTOPLASMIC INJECTION OF dsRNA ON TRANSIENT EXPRESSION	OF THE EGFP GENE MICROINJECTED INTO RAT EMBRYOS
Authors: Takizawa A. , Takahashi M. , Hisamatsu S. , Nakai M. , Shino M. , Sonoki S. , Kashiwazaki N. 
Published: December 2004

RNA interference (RNAi) using double-stranded RNA (dsRNA) is a useful tool to inhibit posttranscriptional specific gene expression in mammalian cells. We investigated the effects of cytoplasmic injection of dsRNAs on transient gene expression in rat embryos when the enhanced green fluorescence protein (EGFP) gene was injected into pronuclei of zygotes. Wistar strain females were superovulated by injections of eCG and hCG and mated with males of the same strain. Pronuclear stage embryos were collected 29 h after hCG injection. The construction of the transgene, the EGFP gene controlled under the CMV-IE promoter, was dissolved at a concentration of 5 μg/mL in DNA injecting buffer (10 mM Tris-HCl, 0.1 mM EDTA), and it (3–5 pL) was microinjected into the pronuclei of embryos. After injection, embryos were cultured in KRB (Toyoda Y and Chang M C. 1974. J. Reprod. Fertil. 36, 9–22) at 37.0°C in 5% CO2 and 95% humidified air until observation. In Experiment I, transient expression of the EGFP gene after microinjection into pronuclei of zygotes was examined. The EGFP expression in the embryos was observed at 6 h intervals until 48 h after the injection using fluorescence microscopy. In Experiment II, the inhibitory effect of dsRNAs targeting the EGFP mRNA was investigated. The dsRNAs for the EGFP were synthesized with T7 RNA polymerase from PCR product amplified with a pair of hybrid primers for the EGFP and T7 RNA polymerase priming site. Then the dsRNAs (1 μg/μL) solution (3–5 pL) was injected into the cytoplasm of the DNA-microinjected embryos 1 h after microinjection. As a control, the TE buffer solution (3–5 pL) was injected into the cytoplasm of the DNA-microinjected embryos. These embryos were cultured and then observed by fluorescence microscopy 48 h after DNA injection. Fluorescent intensity of the embryos was captured and analyzed with image analyzing software (Scion Image, Scion Corporation, Frederick, MD, USA), and all data were analyzed by χ2 test. In Experiment I, the initiation of the fluorescent embryos was observed 24 h after DNA microinjection, and the proportion of fluorescent embryos in five replicates reached maximum (67%, 106/159) at 48 h after DNA injection. As shown in Table 1, there was a difference (P < 0.05) in the rate of fluorescent embryos between the dsRNA-injected group (18.4%, 33/179; range of 3.0–42.3%) and controls (58.8%, 77/131; range of 42.9–100%) in five replicates of Experiment II. The results of the present study suggest that injection of dsRNAs targeting the EGFP mRNA into the cytoplasm had an inhibitory effect on the transient expression of the EGFP gene microinjected into pronuclei of rat embryos. The inhibition system of the present study provides a powerful tool to study specific gene silencing, gene function, and development of early embryos.

Table 1.
Effect of cytoplasmic injection of dsRNA on transient expression of the EGFP gene microinjected into rat embryos