Characterization of Constitutive Promoters for piggyBac Transposon-Mediated Stable Transgene Expression in Mesenchymal Stem Cells (MSCs)

Sheng Wen,Qian Zhang,Hue H Luu,Ningning Wu,Rex C Haydon,Jixing Ye,Fang Deng,Youlin Deng,Lewis L Shi,Hongmei Zhang,Wenwen Zhang,Xian Chen,Yasha Li,Wei Liu,Junhui Zhang,Guanghui Wei,Zhonglin Zhang,Ning Wang,Guolin Zhou,Kang Yang ,Lin Zhan ,Yan Zhang ,Tong‐Chuan He

doi:10.1371/journal.pone.0094397

Abstract

Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.

Highlights

Mesenchymal stem cells (MSCs) are multipotent progenitors and can undergo self-renewal and differentiate into multi-lineages, including osteogenic, chondrogenic, and adipogenic lineages [1,2,3,4,5,6]
In embryonic stem cells (ESCs) the control of the gene expression program that establishes and maintains ESC state is dependent on a small number of master transcription factors as most of the chromatin is in a repression state [13,14,15,16,17]
In order to achieve a high level of transgene expression in mesenchymal stem cells (MSCs), we conducted a comprehensive analysis of six commonly-used constitutive promoters for their abilities to drive reporter genes RFP and luciferase in the stable lines of MSCs and the widely used somatic line HEK-293 cells

Summary

Introduction

Mesenchymal stem cells (MSCs) are multipotent progenitors and can undergo self-renewal and differentiate into multi-lineages, including osteogenic, chondrogenic, and adipogenic lineages [1,2,3,4,5,6]. We systematically analyzed six types of constitutive promoters for their abilities to drive the expression of reporter genes RFP and firefly luciferase in somatic HEK-293 cells and MSC-like iMEF cells. Our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term transgene expression using the piggyBac transposon vectors in stem cells such as MSCs. Materials and Methods

Results

Conclusion