Construction Of Transgenic Vectors And Promoter Analysis Of Zebrafish Somatostatin

Somatostatin (also known as growth hormone-inhibiting hormone (GHIH) or somatotropin release-inhibiting factor (SRIF)) is a peptide hormone that regulates the endocrine system and affects neurotransmission and cell proliferation via interaction with G-protein-coupled somatostatin receptors. Somatostatin has two active forms produced by alternative cleavage of a single preproprotein:one of 14 amino acids, the other of 28 amino acids.In teleost fish, there exists six different somatostatin genes which have been named ss1, ss2, ss3, ss4, ss5, and ss6. The six different genes along with the varified somatostatin receptors allow somatostatin to possess a large range of functions.The zebrafish ssl is expressed in both the central nervous system and the pancreas. Based on the Tol2 transposon found in medaka, a recombinant plasmid was constructed by Gateway technology which contains an ubiquitous promoter, ORF of ssl, EGFP fluorescent reporter gene and a long terminal repeats recognized by Tol2 transponase. We co-injected the plasmid(100ng/ul) and in vitro transcripted Tol2 mRNA(100ng/ul) at a ratio of 1;1 into the plasma of one-two cell stages embryos and the contrast was only injected plasmaid (50ng/ul) and analyse the transient expression pattern. We found that CMV/SP6 ubiquitious promoter can produce embryos with EGFP systemic expression.24 hour post fertilization embryos were picked up and used to analyze the transgenic efficiency. Compared with injection of only constructed plasmids, the EGFP-positive embryos rate of co-injection of Tol2 mRNA is much higher(about 56.79% verse 27.45%). The expression pattern of contrast group.was mosaic while the coinjected group was inclined to be striped; Comared with the coinjection group(34.08%), the ratio of EGFP positive embryosin the contrast group is 5.54%, which shows the plasmid-injected group is more easily quenching.The zebrafish ss4 is mainly expressed in the endocrine pancreas and transiently expressed in the floor plate in early embryos. Bioinformatic analysis of 2467bp of the zebrafish ss4 5’ flanking regions showed that there exists many potential transcript factor binding sites might involved in the process of heptopancrease development and cardiovasclation.2467 bp was cloned and divided into 5 fragments with different lengths, we then subcloned them into pGL3-basic vector and were named as P1,P2,P3,P4 and P5 respectively. The above five plasmids were transfected into HepG2 cells and Promega Dual Luciferase Reporter Gene Assay Kit was used to analyse the promoter activity with different length of promoter fragments. We found that-2200bp upstream of zebrafish somatostatin4 start sites (A+1TG) may exists the core promoter and some positive and negative elements may locate at -500bp and -1000bp to -2000bp respectively.