Production of transgenic animals and determination of transgene and sex by polymerase chain reaction

Abstract

The content will be divided into two main chapters : production of transgenic mice carrying human gastrin/SV40 large T antigen fusion genes, and determination of transgene and sex in single mouse blastomere by polymerase chain reaction. Tissue-specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV40) large tumor (T) antigen. In an attempt to induce the tumors or cancers in stomach tissues, nine transgenic mice were generated that carried a chimeric gene composed of the SV40 T antigen gene fused to the exon ll of a human gastrin gene including $5^\prime$ and $3^\prime$ flanking sequences. It was presented through germ-line transmission that three transgenic mice were mosaic. The transgene was expressed in all tissues examined by RT-PCR whereas endogenous mouse gastrin transcripts were detected only in stomach and brain tissues of the trangenic mouse. The antral region of transgenic mice (TEX-3 line) exhibited abnormal state in appearance although stomach sections of transgenic mice did not yet show any histological or pathological changes. If tumors or cancers will be developed in stomach tissue of transgenic mice, the animal model will be useful for understanding of tumorigenesis and physiology of the organ. In this thesis and efficient method for embryo biopsy was developed. The biopsy method includes the following procedures;embryo holding, partial dissection of zona pellucida, squeezing a single blastomere from an embryo, gentle suction the blastomere into the blunt microneedle, and expelling the blastomere from the blunt microneedle. All of 260 embryos biopsied at 4-cell and morular stage were survivied. Developmental rate of the embryos to normal blastocysts was 93% and 94%, respectively. Blastocysts developed from 4-cell biopsied embryos were smaller than the normal, but were otherwise enentirely typical in appearance. Mean cell number of the biopsied blastocysts were 24.7±5.2 and was reduced more ...