Constrction And Detection Of Porcine Muscle-specific Inducible System

How to get promoters that not only strong specificity and higher efficiency but also safety in the application of transgenic animals becomes a high heat problem with the development of transgenic technology. In this study, the CMV ubiquitous promoter was replaced by porcine muscle-specific promoter a-actin in the mediation of the ecdysone inducible system on the basis of that a higher regulatory and porcine muscle tissue-specific promoter has been isolated by the researchers of our laboratory. And finally we constructed a porcine muscle-specific inducible expression system. The relationship between the expression of the muscle-specific inducible system and the time, as well as the induction dose was verified at the cellular level. An experimental basis for gene therapy and the tissue-specific expression of exogenous gene in skeletal muscle were provided in this study, and laid a foundation for further study of transgenic pigs in the same time. The main findings are as follows:1. This study was based on the ecdysone inducible system, we removed the constitutively active CMV promoter of pVgRXR and replaced it with a porcine skeletal muscle a-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. In order to detect if the foreign gene can be successful expression in this inducible system. The EGFP gene was used as a reporter gene to detect whether the new designed expression system could expresses foreign genes, and finally it was defined by the experiments successfully.2. We replaced the reporting gene EGFP by β-galactosidase gene which was used to detect the expression of the system in the above-mentioned system. Results show that the expression of the reporting gene and both the time and the induction of the induction dose was in proportion in a certain range. Gene expression reaches the maximum when the induction time is72h and induction dose is36μM. We found that the expression level of the system in the differentiated C2C12cells (myotubes) was significantly higher than undifferentiated C2C12cells(p<0.01). Proved that the system can be specifically expressed in myotubes. 3. We transfected our newly designed expression system into the mouse C2C12muscle myoblasts, and then found five postive clones, and established stable cell lines via the selection of G418. And it will been used for the following studies.4. Designed a pVg-actin/pIND-MyoD muscle-specific inducible system for porcine MyoD gene, and transfected it to differentiated C2C12cells. It fully demonstrated the high inducible abilitly of the muscle-specific inducible system by the proof that the expression of MyoD was17fold higher than the control group under the condition of36μM and48hours.