| The technology of transgenic from generation to now has gone through decades of development, especially with the rapid development over the past decade. However, the ultimate aim of transgenesis is not to produce several transgenic animals,but to service for the needs of human.Objective: In this experiment, the insulin-like growth factor-I transgenic wool sheep that were cultured by our laboratory and their descendants were used and IGF-I gene was excepted for the research objectives.Through the IGF-I integration sites, exogenous gene copy number, transcription level and translation level of the transgene wool sheep were analyzed in order to provde a reference for the effcient production of transgenic wool sheep and lay the foundation for the function of the exogenous gene.Methods: Experiment one: The wool wool sheep and their offspring which were constructed by our laboratory were identified which one was IGF-I transgenic wool wool sheep. According to the exogenous gene plasmid sequence, specific primers were designed. The PCR method was used to identify positive transgenic wool sheep which were constructed in this laboratory. Experiment two: Using real-time PCR technology, design real-time PCR primers for identifying positive transgenic wool sheep exogenous gene copy number. Experiment three: The real-time PCR was used to analysis IGF-I m RNA expression levels of transgenic wool sheep skin tissue. Expression four: Western blot was used to analysis protein expression of exogenous gene in transgenic wool sheep skin and visceral, detect translation level of exogenous gene. Expression five: Using chrimosome walking technique, the transgenic wool sheep genomic DNA was extracted,then analysis the integration site of exogenous gene.Results: Expression One: the results showed: 0152, 0016, 118, 0217 were IGF-I transgenic wool sheep, respectively. Expression Two: the results showed: copy number of the IGF-I transgenic wool sheep 0152, 0218, 0217, 0216 were 3, 1, 1, 1, respectively. Expression Three: the results showed: the IGF-I gene relative expression of 0152,0016,118,0217 skin tissue were 2.87 times, 2.03 times, 1.86 times and 1.85 times compared to the control group, and the differences were highly significant(P <0.01). IGF-I gene relative expression of primary wool sheep 0152 skin tissue were 2.07 times and 1.74 times compared to 118 and 0217, the differences were highly significant(P <0.01); IGF-I gene relative expression of primary wool sheep 0016 skin tissue were 1.789 times and 1.50 times compared to 118 and 0217, the differences were highly significant(P <0.01); the relative expression level in primary wool sheep 0152 skin tissue of IGF-I gene compared to 0016 was not significantly different(P> 0.05); the relative expression level in generation wool sheep 118 skin tissue of IGF-I gene compared to 0217 was not significantly different(P> 0.05). Expression Four: the results showed: the IGF-I protein relative expression of 0152,0016,118,0217 skin tissue were 1.55, 1.08, 0.55, 0.62, and were the same age ordinary wool sheep 213%, 187%, 129%, 127%,respectively, the differences were significant(P <0.01), IGF-I protein expression in skin tissue of IGF-I transgenic wool sheep were compared with ordinary wool sheep improved significantly. Expression of IGF-I protein in the internal organs of IGF-I transgenic wool sheep 0152 compared with ordinary, the rasults showed that the relative expression of IGF-I protein in transgenic wool sheep heart, liver, spleen, lung, kidney, muscle, skin were 0.348, 2.571,0.863,0.731, 0.316, 0.124, 0.481, respectively, the relative expression of IGF-I protein in ordinary wool sheep heart, liver, spleen, lung, kidney, muscle, skin were 0.357, 2.564, 0.847, 0.727, 0.304, 0.127, 0.215, respectively. The IGF-I protein relative expressoon level of 0152 skin tissue are 223.8% compared to ordinary wool sheep, Significant differences(P <0.01), the relative expression of IGF-I protein in the rest of tissues between transgenic wool sheep 0152 and ordinary wool sheep were not significantly different(P>0.05). Expression Five: the results showed: the chromosome walking techniques was used to get the foreign gene insertion sites Located in the genome on chromosome 18 between 7097202-7097206. Conclusion: According to analysis genetic stability of the IGF-I transgenic wool sheep, the results showed that exogenous was successfully integrated into the genome of wool sheep, and with the host genetic stability to thire offspring. Exogenous gene at the transcriptional and translational levels in transgenic primary wool sheep and generation wool sheep can be stably expressed, and its expression in skin tissue specificity. |
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