In vivo amd in vitro development of transgenic rabbit embryos

• Main Authors: Chien-Hong Chen, 陳建宏
• Other Authors: Jyh-Cherng Ju
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/90217090537164426337

碩士 === 國立中興大學 === 畜產學系 === 91 === Transgenic rabbits have been used as both an animal model and the bioreactors for pharmaceutical purposes. Among the methods to generate transgenic animals, pronuclear microinjection and nuclear transfer (NT) are the most popular techniques. In this study, the effects of promoters on the expression of foreign genes and embryo development were evaluated. Nuclear and cytoskeletal reorganization of the nuclear transferred embryos were also examined after transfer of gene-transfected somatic cell nuclei. In Experiment 1, the enhanced green fluorescent protein (EGFP) genes, driven by different promoters, were introduced into rabbit zygotes. The blastocyst rate was significantly lower in the zygotes injected with the gene driven by CMV promoter than by β-actin promoter (47% vs. 68%, p < 0.05). Expression of EGFP in the blastocyst had no significant difference between two treatment groups, but more embryos with mosaic pattern of EGFP expression were observed in the the CMV promoter driven group. This result indicated that CMV promoter negatively affected the in vitro development (IVD) of rabbit embryos. In the Experiment 2, the effects of different foreign genes on IVD of embryos following injection were investigated. After gene injection, percentages of blastocyst formation were 45 and 40% in the groups injected with hepatitis B surface antigen (HBsAg) and human serum albumin (HSA) genes, respectively, which were both significantly lower than that in the control group (76%, p < 0.05). The result suggested that injection of foreign genes was detrimental to the IVD of rabbit embryos. However, no reduction in total cell numbers and no increase in the ratios of apoptotic cells of the blastocyst from the HBsAg gene-injected embryos were detected. Tweenty-five pups, regardless of the embryos injected with HBsAg gene, HSA gene, or co-injection of HSA with EGFP genes, were born after embryo transfer and five of them were verified as transgenic pups by PCR analysis. Changes in the nucleus and the cytoskeleton were evaluated after parthenogenetic activation and NT in the final two experiments (Experiments 3 and 4). Developmental abnormalities, including aberration and alteration in both the chromosome and the spindle, were observed in the reconstructed embryos. Presumably, these changes would cause the failure of subsequent development of the reconstructed embryos. In addition, ear skin fibroblasts derived from a male rabbit were transfected with the EGFP gene and the developmental competence of reconstructed embryos was evaluated following NT. Results showed that no significant differences were observed in the rates of morula/blastocyst development (14% vs. 13%, p > 0.05) between these two types of donor cells (transftected vs. non-transfected), and the expression of EGFP gene in the reconstructed embryos was 46% when developing into the morula and blastocyst stage after in vitro culture.