Inhibition of angiogenin gene expression of mouse embryos by RNA interference

• Main Authors: Shih-Kuan Wang, 王世寬
• Other Authors: Pin-Chi Tang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/41124693026615283451

碩士 === 國立中興大學 === 畜產學系 === 93 === RNA interference (RNAi), mediated by double-stranded RNA (dsRNA), has been demonstrated being capable of silencing gene expression in a sequence-specific manner. It has been applied to study the function of genes interested. Mouse angiogenin (Ang) is a constitutive protein in the blood and RNase A gene family including Angiogenin, RNase 1 and RNase 4 are located on mouse chromosome 14. The aim of this study was to investigate the effect of Ang expression on the development of mouse embryos by RNAi. In Experiments 1, the expression patterns of RNase A gene family in the pre-/postimplantation mouse embryos and different mouse cell lines were examined by RT-PCR. The expression of Ang was detected at E1.5, E2.5 and E3.5, E7.5, E9.5 and E12.5 mouse embryos/fetuses. RNase 4 was expressed weakly in E7.5 fetus but expressed strongly at the stage beyond. However, RNase 1 could only be detected in E9.5 and E12.5 fetuses. Ang and RNse 1 were detected in mouse CT-26, 3T3 and B16F10 cell lines, but RNase 4 was not expressed in B16F10 cells. After transfection of the three Ang-RNAi expression vectors (pUC-ANG1, -ANG2, -ANG3) into CT-26 and B16F10 cells, the expression of Ang was inhibited significantly (P<0.05). However, different inhibitory efficiencies were observed in different cell lines. In CT26 cells, the expression of Ang was reduced to 22%, 47% and 47% by pUC-ANG1, pUC-ANG2 and pUC-ANG3, respectively, while in B16F10, they were 57%, 18% and 33%. Additionaly, the inhibitory effects of pUC-ANG2 and pUC-ANG3 to Ang expression in B16F10 were significantly higher than pUC-ANG1 was. In Experiment 2, the U6-ANG1 RNAi expression cassette was introduced into the pronucleus of mouse zygotes by microinjection to evaluate the effect of Ang on the development of mouse embryos. Results showed that there was no significant difference on the percentages of blastocyst formation among the control (non-injected), U6-ANG1 injected, and U6-ANG1+EGPF coinjected groups. The percentages of embryos developed to morula stage in the U6-ANG1-injected (65%) and the coinjected groups (58%) were significantly lower than that of the control group (73%). A significant difference was also found between the two gene-injected groups. After transfer of the coinjected embryos into recipient mice, four out of the twenty four pups born were verified as EGFP transgenic mice by PCR, but none of them carried the U6-ANG1 insert. In this study, Ang expression was detected in the pre- and postimplantation mouse embryos And the Ang-RNAi expression vectors could inhibit the expression of Ang in different cell lines. Introduction of siRNA targeting Ang gene expression to mouse zygots might be lethal to the postimplantational development of mouse embryos, which requires further investigation.