| During decades of swine breeding, improvement of lean meat percentage instead of muscle quality has been attached great importance both at home and abroad. However, the increase of intramuscular fat can improve tenderness of muscle, succulence and taste. Our laboratory construcred a muscle specific expression vecto,which is pGL3-MyoG-PPARγ-Flag-poly(A) Signal, including the muscle specific gene myogenin(MyoG) promoter of swine and fat related genes peroxisome proliferator-activated receptorsγ(PPARγ),linear processing after verification in C2C12 cells, pronucleus microinjection to successfully prepare MyoG-PPARγ transgenic mice so as to conduct test and verification in vivo. The research basically verifies tissue’ specific promoter’s influence on MyoG-PPARγ transgenic mice and activity of Myo G promoter at MyoG-PPARγ transgenic mice phenotype, DNA level, mRNA level, protein level and finally proves that muscle specific promoter MyoG can drive exogenous PPARγ gene’s up-regulated expression in vivo.Currently, the main research results are as below:1. Four founder mice are used for reproduction and system building. Founder ♂1 mouse has stably-inherited offspring, and so far its gene has been inherited to F5 generation. Positive rate of founder ♂1 transgenic mice is over 95%, reaching the standard for system building.2. Drawing the weight growth curve of mice from 1-12 month old, and detecting fat-related indexes in mice serum of MyoG-PPARγ transgenic mice and FVB wild type in their three stages(one-month old, two-month old and three-month old). The result reveals that the weight growth between MyoG-PPARγ transgenic mice and FVB wild type mice has no outstanding difference at all stages(p > 0.05); the difference of both total cholesterol and triglyceride content in the serum are evident for MyoG-PPARγ transgenic mice and FVB wild type in one-month old, two-month old(p < 0.05). As indexes of each detected mouse are within the normal scope of FVB mice, it can be confirmed that the insertion of exogenous gene won’t affect the health of transgenic mice.3. Employing absolute quantification PCR technology to collect data concerning the copy number of MyoG-PPARγ transgenic mice at DNA level. Data suggests that copy number of founder Myo G-PPARγ ♂1 mice is 2.24 and exogenous gene is not single-copy insertion. By counting the copy number from random F0-F5 generation transgenic positive mice, it is found that with the increase of generation, exogenous gene copy numbers of MyoG-PPARγ transgenic mice are increased for a majority of offspring despite a minority which see a decrease.4. Using qRT-PCR technology to measure expression quantity of exogenous gene PPARγ of one-month old, two-month old, three-month old MyoG-PPARγ transgenic mice as well as their expression quantity of downstream target genes A-FABP and LPL mRNA, it is found that MyoG-PPARγ transgenic mice see significant rise in the aforementioned items compared with FVB wild type mice(p < 0.05). By longitudinally comparing the expression quantity of exogenous gene PPARγ of MyoG-PPARγ transgenic mice at one-month old, two-month old and three-month old, it is revealed that the expression quantity of one-month old MyoG-PPARγ transgenic mice increases the most compared with that of the exogenous gene PPARγ of FVB wild type mice.5. When using western Blot, immunohistochemistry technology to detect the change of exogenous gene PPARγ protein level, the result shows that the expression quantity of PPARγ protein in MyoG-PPARγ transgenic mice is higher than that of the FVB wild type mice.6. When detecting the triglyceride content in the leg muscle of one-month old, two-month old, three-month old MyoG-PPARγ transgenic mice and FVB wild type mice, it is found that the expression quantity of MyoG-PPARγ transgenic mice triglyceride is significantly greater than that of the FVB wild type mice(p < 0.05) during the whole growth stage. As mice grow, their intramuscular fat(IMF) deposition also gradually increases. |
PDF Full Text Request