Tuesday, August 3, 2004
10:30 AM–12:30 PM
(546) TARGETING ANGIOGENIN GENE EXPRESSION IN EARLY MOUSE EMBRYOS USING SHORT DOUBLE-STRANDED RNAS.
Wang, Shih-Kuan1, Fang, Jung-Da1, Liao, You-Di2, Tang, Pin-Chi1, Ju, Jyh-Cherng1, 1 Department of Animal Science, National Chung-Hsing University, Taichung, Taiwan2 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
ABSTRACT- In addition to the angiogenic and ribonucleolytic activities, angiogenin has been suggested to involve in other biological functions in a variety of cells. In this study, we investigated the effects of angiogenin gene expression on the development of mouse embryos by RNA interference (RNAi). Experiment 1 was conducted to investigate the expression of angiogenin during mouse embryogenesis. Mouse embryos/fetuses were collected at Day 2.5, 3.5, 7.5, 9.5, or 12.5 post coitus and total RNAs were extracted and analysed using reverse transcriptase polymerase chain reaction. Our results showed that the expression of angiogenin could be detected at the morula stage (Day 2.5) and onwards. Experiment 2: Three different regions of DNA sequences from mouse angiogenin gene (NCBI gene bank, access no. NM007447) were constructed into pUC19 plasmids and designated as pUC-ANG1, -ANG2, and -ANG3, respectively, for production of short interfering RNAs (siRNAs). The DNA clones were transfected into CT-26 cells by polyethylenimine. Results showed that two of these three clones, pUC-ANG1 and -ANG2, suppressed the expression of angiogenin mRNA efficiently. Experiment 3: The DNA pUC-ANG1, targeting angiogenin transcripts, was introduced into mouse zygotes by pronucleus microinjection. After 3 days of in vitro culture, no significant difference was observed in blastocyst formation between the TE buffer-injected and DNA-injected embryos (P>0.05). In conclusion, specific sequences of DNAs could effectively block angiogenin expression in CT-26 cell line. It also suggested that angiogenin gene expressed in early mouse embryos might be dispensable in the development of preimplantational embryos. Further studies are required to clarify the functions of angiogenin during mouse embryogenesis.
KEY WORDS: pronucleus microinjection, mouse, angiogenin, rna interference