Identification Of The Core Promoters Of Growth Hormone Genes And Their Species-specific Regulation In Pigs And Mouses

Growth hormone is a core factor of the growth axis in the regulation of growthand development, having a very wide range of regulation of cell growth and materialmetabolism for animal tissues, significantly promoting the growth of cartilage, boneand other organizations, stimulating the synthesis of collagen and protein, andpromoting the uptake and utilization of cell cycle amino acids organization forrelated tissues.These features of the growth hormone make it the first gene being used in thetransgenic animal research. Till now, GH transgenic offspring has been successfullygenerated in poultry, rodent, fish, insect, pig and so on. The growth rate, feedefficiency and carcass lean meat percentage is substantial increased in the GHtransgenic pigs, compared with non-transgenic ones. However, there are still somesequelae existed, such as in the G0transgenic pigs, still born and malformation rateis somewhat high.To overcome the above described problems and avoid the side effects inducedby the expression of exogenous growth hormone, thus the study on GH expressionregulation mechanism and exploration its role in vivo, is important for livestockproduction and clinical application. However, lots of reports are about studies on thetranscriptional regulation of human and rat growth hormone, but the study on theexpression regulation mechanism of mouse and porcine growth hormone has notbeen reported.Early study proved that cell type-specific restriction of rGH requirescombinatorial efforts of Pit1, TR, and an unknown factor that recognizes-161/-146 site together with the corepressor machinery including N-CoR. The-500bppromoter of human pituitary GH (hGH-N) gene contains binding sites for Pit1, Sp1,and zinc finger proteins. However, it is not sufficient for efficient expression intransgenic mice and a locus control region (LCR), located14.5to32kb upstream, isrequired for efficient and position-independent expression of the hGH-N transgene.This study was firstly focused on the structure of pig and mouse GH genepromoter, using bioinformatic method to compare and analyze their5’flankingsequences. A series of promoter deletion vectors were built, and dual luciferasereporter gene activity analysis was used to detect the activities of different lengthfragments of the GH gene promoter in pituitary cells and non-pituitary cells. Andthen the structure of pig and mouse GH promoter region was analyzed and compared,and the differences on the transcriptional regulation of the two species were alsodetermined. Trans-acting factor analysis was performed to the GH promoter regionusing the TESS website. Utilizing Sp1inhibitor Mithramycin A, the Sp1wasconfirmed to play an important role on the regulation of GH transcription. The Pit-1over-expression vector and site-directed deletion vector were used to explore the roleof Pit-1to GH cell-specific expression regulation and determine its binding sequence.The results show that the Pit-1was the main factor in the control of porcine GHcell-specific expression, but it did not largely affect the activity of the promoter ofmouse growth hormone. In addition, recently research has shown that cytokinessecreted from lymphocytes can enhance the transcriptional activity of human growthhormone. Therefore, the cell culture experiment was performed and the resultsshowed that the cytokines IL-6and IL-11were significantly positive on theregulation of pigs and mouse growth hormone.