| As a a regulatory system of inducible expression,Tet-on regulatory expression system has been applied to biogenic reactions which become an effective tool to study the function of genes,especially in mice.It has become a very effective induction model to study the gene function of transgenic animals.Growth hormone is naturally produced by the pituitary gland,a pea-sized organ at the base of the brain,which can promote maturation,improve percentage of lean meat and ameliorate feed conversion rate of different species,but there are also side effects.This may be due to overexpression in transgenic animals,and the exogenous GH gene has a biological effect from the embryo to adult,and the abnormal development of the organism is likely to be related to the autocrine abnormality of GH in multiple tissues.The concentration of doxycycline(DOX)was positively correlated with the level of exogenous gene expression in a certain range;therefore,expression level of exogenous gene was changed by controlling the concentration of tetracycline and DOX.It was helpful to grasp the transgene expression level at different levels and different growth stages,understand its role in animal growth and development stage,and further study the biological function.This study aims to explore the construction of eukaryotic expression vector carrying porcine growth hormone gene(pGH)by using molecular biology method.After transfected into PIEC cell lines(Porcine iliac artery endothelial cells)through cationic liposome successfully,the efficiency and expression level of inducible expression(Tet-on regulatory expression system)after adding DOX were verified at the cellular level.Then we used microinjection method for preparing transgenic mice and conducted a preliminary study on their functions.The results were showed as following:First,construction of recombinant expression vector(pTTGH)was carried out successfully.On the basis of the successful construction of recombinant vector pTRE-GH12,pTRE-GH fragment was amplified by PCR from pTRE-GH 12,then the ligation of Sal I digested fragment to Sal I digest vector pCAGGS-rtTA,the recombinant vector pCAGGS-rtTA-TRE-GH12(pTTGH)was construted successfully.The pTTGH vector DNA was transfected PIEC cells by way of Cationic liposome.After enriched by genetic resistance agent(G418),Quantitative fluorogenic real-time PCR(RT-qPCR)assay for pGH mRNA expression level when doxycycline(DOX)was added to the culture medium for induction.On the other hand,different concentrations of doxycycline prepared were added into the cell culture medium,RT-qPCR and Western Blotting assay for determining the expression efficiency of porcine growth hormone.The results of digestion,sequencing and restriction showed that the pTTGH vector was constructed successfully and could meet the requirement of the next experiment.RT-qPCR results showed that expression of mRNA of porcine growth hormone was increased 4.21,6.79,73.27,34.29 and 30.07 times when DOX adding into cell culture fluid after 12,24,36,48 and 60 h and compared with the control group(no DOX).Inducied with adding different DOX concentration of 100,200,300,400,500,600,700,800,900 and 1000 ?g/mL in culture medium,the expression of nRNA of porcine growth hormone were 9.87,11.45,26.87,30.09,36.77,47.84,46.87,41.23,35.27 and 29.87 times than' the control group(no DOX),and the difference was extremely significant(P<0.01).Results of gray intensity analysis(Western Blotting)showed that relative expression of protein of porcine growth hormone were 0.70?0.68?0.62?0.71 and 0.72,when doxycycline adding into cell culture fluid after 12,24,36,48 and 60 h compared with the control group was 0(no DOX).The results showed the vector has been constructed successfully and which can realize controllable expression of pGH.This study established a foundation for preparing for controllable expression of pGH in the transgenic animal and helps us to further study the function of growth hormone.The preparation of pTTGH transgenic mice was transfected through prokaryotic microinjection method.Based on Southern blot results,positive founders were bred for getting offsprings.When get the offsprings of transgenic mice,the induction experiment was carried out with the induction substrate doxycycline DOX in the drinking water of the transgenic mice at 3-10 weeks old.The results showed that the body weight of transgenic mouse continued to increase during the induction period,and the growth rate is higher than that of non transgenic mice and transgenic control group(no DOX),the level of pGH in serum of transgenic mice was significant higher than that of control mice(P<0.05).The expression level of endogenous IGF-1 in serum samples was detected by ELISA.The results showed that the level of IGF-1 in the serum of transgenic mice was obviously increased,indicating that exogenous GH regulates the growth of the organism by regulating the expression of IGF-1.The morphological and pathological changes of the heart,liver,spleen,lung,kidney,dorsal muscle and leg muscle of transgenic mice were observed,the results of HE showed that functional organs of the heart,liver,spleen,lung and kidney were no visible lesions of transgenic mice after adding antimycin DOX via drinking water,compared to wild-type mice sibbing with DOX.Meanwhile we also get the conclusion the foreign gene pGH has already integrated into mouse into mouse chromosome,and it can inherit in accordance with the laws of Mendelian inheritance.Compraed with the control group,there was no harm cause to the organism of offsprings of transgenic mice in vivo by the the expression of exogenous growth hormone after incuced by the DOX and the healthy condition of transgenic mice were not significantly different.The establishment of modle of pGH transgenic mouse contributed to explore the influence of the molecular mechanism and signal pathway of growth hormione secration.Meanwhile it would promote the application of GH in livestock production intensely;it also provides a theoretical foundation and technical support for the preparation of safe and efficient GH transgenic livestock.By using technology of isobaric tags for relative and absolute quantitation(iTRAQ),6 samples of brain,liver and muscle in transgenic mice and non-transgenic mice were used for mass spectrometry identification,and then the bioinformatics analysis was carried out.Then the effect of GH transgenic mice on the body was identified,which will further provide theoretical basis for the overexpression of GH on the molecular mechanism of brain development and energy metabolism.The result of functional enrichment analysis of differentially expressed proteins from brain tissues illustrate that a total of 360 DEPs were detected from transgenic and non-transgenic mice brain,148 highly expressed proteins and 242 low expressed proteins of transgenic group;a total of 170 DEPs were detected from transgenic and non-transgenic mice liver,74 highly expressed proteins and 96 low expressed proteins of transgenic group;a total of 390 DEPs were detected from transgenic and non-transgenic mice muscle,156 highly expressed proteins and 234 low expressed proteins of transgenic group.Through comparative analysis,we can come to conclusion that the effect of excessive GHacted on brain,liver and muscle after overexpressing GH.We found that differential proteins by GO analysis and K.EGG pathway analysis,which mainly enriched in the process of RNA shear,intercellular protein transport and mRNA process-cycle and insulin signaling pathway,the AMPK pathway,the PI3K-Akt signaling pathway,and the PPAR signaling pathway. |
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