Low levels of the reverse transactivator fail to induce target transgene expression in vascular smooth muscle cells.

Nikenza Viceconte,Tomás Mckenna,Maria Eriksson,Carlos E Ambrósio

doi:10.1371/journal.pone.0104098

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.

Highlights

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disorder characterized by multiple features and pathologies typical of advanced age
The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina located beneath the inner nuclear membrane [3]
To analyze the transgene expression at the RNA level, we used RT-PCR with primers that were specific for human lamin A, lamin Adel150, and the reverse transactivator (Figures 1A–1C)

Summary

Introduction

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disorder characterized by multiple features and pathologies typical of advanced age. The LMNA c.1824C.T, p.G608G mutation and other lessprevalent mutations, including the LMNA c.1822G.A, p.G608S mutation [3,4], result in the partial activation of a cryptic splice site and in the removal of the 150 carboxy-terminal nucleotides of exon 11 [3,4]. This internal deletion leads to the expression of a truncated lamin A protein with an internal deletion of 50 amino acids, called progerin [3,5,6]. The expected frequency for each individual genotype was 25%. doi:10.1371/journal.pone.0104098.t001

Objectives

Methods

Results