Environmental Risk Assessment Of EGFP Transgenic Mice

In recent years, transgenic animal technologies have been developed rapidly and been used widely in many fields, such as food, medicine, agriculture and biomaterial. The security issues of transgenic animals and their products and environmental safety are increasingly emerged. However, The researches related with environmental risk assessment of genetically engineered animals was few.In this paper, the eGFP (enhanced green fluorescent protein) transgenic mice were as animal model.(1) the transgenic mice were passaged with single-line method (founder:F0,6; F1, the offsrings of FO and wild Kunming mice,8; F2, the offsrings of F1,19; F3, the offsrings of F2,22; and F4, the offsrings of F3,39). We used PCR and Fluorescent Protein Observation system to detected the exogenous genes of these mice.(2)The relative time quantitative PCR (RT-QPCR) method was performed to detect exogenous genes expression in the tissues of mice.(3)Then we evaluated the impact of genetically engineered gene on the environment with a total of twelve4and8week-old full-sib mice (treatment:control=6:6, and the same size of male and female mice). The total aerobe, total anaerobe, main beneficial bacteria (including Bifidobacteria, Lactobacilli, Bacteroides, and Streptococci), main harmful bacteria (including Coliforms, Salmonellae, Enterococci, Clostridia and Staphylococcusaureus) as indicators to detect the changes of microbial flora habiting in various parts of feces of the above two groups of mice were tested.The probability of exogenous eGFP gene impact on the microbial communities in mice gut were tested.(4)At last, PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method were taken for comparison the fecal microbes of transgenic mice and control mice. About3016S rDNA samples from gut microbes in4week-old mice, and29in8-week-old mice were cloned and sequenced. The main results were as follows: 1.The genetically engineered mice had bred four generations, there were a total of94offsrings. The results showed that there is no significant difference between PCR detection and the observation of green fluorescence traits by the X2test, X2=2.7163, X2<X20.05(4), P>0.05. The PCR method is feasible for detection of genetically modified mice. And single-line method is feasible for transgenic founder expandation.2.The total RNA of heart, liver, spleen, lung, kidney, cecum and testis of the mice were extracted and the expression of eGFP and WRPE genes was analyzed with Real-time PCR. The results showed that the exogenous genes expression in heart, liver, spleen, lung, kidney, cecum and testis of the transgenic mice. The expression level in founder tissues was different. But no significant difference was found among the heart, liver, kidney and testis (P>0.05), and there was significant different (P<0.05) between in spleen and cecum. The expression lever of eGFP gene was significantly different in the liver tissue from different generations (F0, F1, F2, F3and F4)(P <0.05), and it was highest from the founder. And the was no significant different(P>0.05) from F1-F4generations. About WRPE gene, similar expression patterns were found.3. We evaluated the impact of genetically engineered mice on the environment with4and8week-old full-sib positive and nagitive transgenic mice offsprings. The results showed that the exogenous genes were not detectable by polymerase chain reaction (PCR) in the manure extracts r or external surrounding soil. Microbial flora of total aerobe, total anaerobe, Bifidobacteria, Lactobacilli, Bacteroides, Streptococci, Coliforms, Salmonellae, Enterococci, Clostridia and Staphylococcus aureusin of transgenic mice were not significantly different from those of control (P>0.05), whether male or female, at4or at8week-old.4.The was no significantly difference of the microbial communities in mice gut as assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) or by16S rDNA sequencing between positive and nagitive transgenic mice offsprings. Furthermore, the phylogenetic analyses showed that the manure bacteria sampled during each of the two stage belonged primarily to three groups, Firmicutes, Bacteroidetes and Actinobacteria. And no unknown microbial flora were found in the mice manure.