SELECTED SEQUENCES OF PORCINE ELONGATION FACTOR 1 ALPHA PROMOTER LEAD TO SIGNIFICANT UPREGULATION OF TARGET GENE RESPONDING TO HUMAN SERUM INDUCTION IN PORCINE CELLS

Abstract

Transgenic pig has been well known as a model and a potential source of cross-species organ transplantation. Fully and partially virus-originated promoters, CMV and CAG were predominantly used for generation of transgenic pigs. In this study we carry out to develop regulatory sequences originated from pig deriving efficient expression of target gene. We selected sequences according to analysis using AliBaba2.1 program, and synthesized the regulatory sequences equivalently to elongation factor 1 alpha (pEf1α) and actin (pAct) of pig, leading to construction of luciferase expression cassettes. Luciferase assay was showed that pEf1α promoter lead to significantly higher expression than CAG in ear skin fibroblasts (pEfb) and to similar expressions in porcine kidney cells pK15 and endothelial cells (pAec) compared with CAG. The pAct promoter was shown to be inefficient expression of luciferase compared with CAG and pEf1α in pEfb and pK15 cells. To inquire into that pEf1α promoters could spontaneously induce gene expression responding to rejection mechanisms by xenotransplantation, we treated normal human serum to pAec. Consequence was pEf1α promoter lead to significant upregulation of luciferase expression, while the level of endogenous pEf1α expression was not changed. We additionally constructed HLA-E and beta macroglobulin(b2m) expression cassettes in conjunction with pEf1α and CAG promoters, and transfected the cassettes into pEfb. We obtained stable cells by neomycin selection for 14 days, and subsequently analyzed HLA-E expression up to 30 days. We were able to observe that HLA-E expression by pEf1α promoter was consistently sustained for 30 days, but it by CAG promoter was dramatically downregulated from 18 days’ later selection. Taken together, we suggest that selected sequences of pEf1α gene in this study were able to regulate stably spatial and temporal expression of target gene in transgenic pig, and to induce distinct upregulation of the gene on rejection incurred in primate xeno-grafted of the pig. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No PJ01384202)” Rural Development Administration, Republic of Korea.