Cloning and Functional Analysis of the Porcine Growth Hormone Gene Promoter

[Objective] This study aimed to clone the porcine growth hormone gene promoter and determine the core promoter sequences and the cis-acting elements. [Method] Sequence of the 5＇flanking region of porcine growth hormone gene was searched out and downloaded from the NCBI website. According to the targeted se- quence, primers were designed and synthesized for the PCR amplification. The 1 882 bp （-1 821 bp-＋61 bp） fragment was amplified by PCR. Nine promoter frag- ments with different lengths were obtained by genome-walking deletion method and then cloned into luciferase reporter vectors. Relative transcriptional activities of these 5＇ terminal-deleted plasmids in pituitary and non-pituitary cells were determined by transient transfection of the rat pituitary adenoma cell （GH3）, porcine lilac endotheli- um cell （PIEC） and porcrne Kidney-15 （PK15） with the constructed dual-luciferase vectors. [Result] Result of DNA sequencing showed that the 1 882 bp fragment of GH 5＇ promoter was successfully cloned. Nine luciferase reporter gene plasmids were constructed. DuaI-Luciferase reporter assay indicated that the promoter inserted into reporter gene vector had very strong cell specificity. [Conclusion] Porcine growth hormone gene specifically expresses in pituitary cells. The minimal promoter of the porcine growth hormone gene is mapped at the region -110 bp-＋61 bp. Promoter regions 218 bp--110 bp and -429 bp--218 bp contain positive regulatory elements.

Construction of Eukaryotic Expression Vector for Pig Ghrelin Gene

[Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pig Ghrelin mRNA sequence published in Genbank. Total RNA was extracted from the small intestine tissue of 13/17 Robertson translocation heterozygous pig, and then was purified and used as the template in later RT-PCR reaction to amplify the full-length pig Ghrelin gene. The correct pig Ghrelin gene fragment was cloned into the pMD19-T simple vector for sequencing analysis. The obtained full-length cDNA of pig Ghrelin gene fragment was digested with both Nhe I and Hind Ⅲ, and then was linked into the eukaryotic expression vector pEGFP-N1 to obtain the recombinant plasmid pEGFPGhrelin. The recombinant plasmid was transected into the fibroblast cells to detect the fluorescence labeled gene expression. [Result] The nucleotide sequence extracted from 13/17 Robertson translocation heterozygous pig was the same as expected; and the eukaryotic expression vector pEGFP-Ghrelin was successfully constructed. [Conclusion] The eukaryotic expression vector constructed in this study can be further used in research on transgenic pigs, but also lays foundation for research on the regulatory mechanism of Ghrelin gene.