The Research Of Safety Assessment On Transgenic Animal By Transgenic Mice Model

In recent 30 years, with the rapid development of the transgenic technique, production of transgenic animal not only brought revolutionary changes to agriculture and biomedical sciences, but also caused a series of related problems such as the safety of transgenic food, ethical issues of transgenic animals, ecological risk and possible environment's problems of transgenic animals and so on., those problems forced people to strengthen management of transgenic animal and enact consummate laws and sign regulations about transgenic animal and transgenic food safety in the world. However, the enactment of laws and regulations needs sound transgenic detection systems to provide technological support. This thesis mainly studies on the detection for transgenic components in mice.1) Detection of positive transgenic miceDNA was extracted from mouse tails by one step phenol– chloroform method, and high quality DNA that OD value between 1.7-1.9 was obtained. We detected positive mice with polymerase chain reaction (PCR) technique, and validated the PCR results with Southern blot. The results showed that three of the four detected mice were transgenic positive, and one has no signal, by PCR and southern blot, while showed positive when detected by fluorescent quantization PCR and TAIL-PCR latter.2) Detection of exogenous gene constructsWe established multiplex PCR method: it is a variant of PCR in which four loci are simultaneously amplified in the same reaction, such as endogenous gene GAPDH, CMV promoter, polyA and exogenous gene of transgenic constructs. We obtained perfect specific amplification results by optimizing amplification system and conditions, which indicated that multiplex PCR can markedly boost efficiency and speed of transgenic detection.3) Detection of exogenous gene integration siteBased on the 5′end of foreign gene, three nested specific primers were designed, together with the short arbitrary degenerate primers which were designed according to the Coden Usage of mouse; genomic fragment flanking the insertion site of the foreign gene was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and sequenced, the homologue blasting results showed two integration sites were identified, one was the tenth intron of Sash1 gene on X chromatosome of mouse, and another is the intergenic spacer region between Nxph1 and AA545190 of VI chromatosome.4) Detection of copy numberThe mouse housekeeping single copy gene B2M, served as an internal control to calculate transgenic copy number. According to the sequences of TMEM66 and B2M, two sets of primers and TaqMan probes were designed to establish a TaqMan real-time PCR assay, and draw a relative quantitative standard curve. The correlation coefficient of standard curve was 0. 990 and 0.985 respectively, the copies numbers of the four founder mouse are 623, 3, 22, and 10 respectively.