In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

Bradley K. Wacker; Lianxiang Bi; David A. Dichek

doi:10.3791/56982

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Method Article

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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DOI:

10.3791/56982

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May 6th, 2018

Bradley K. Wacker¹ , Lianxiang Bi¹ , David A. Dichek¹

¹Department of Medicine, University of Washington

Summary

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Abstract

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

Introduction

The goal of this method is to introduce a transgene into the endothelium of rabbit common carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product both in normal arteries and in rabbit models of human arterial disease. Overexpression of the transgene in disease models can reveal whether the transgene (and its protein product) show promise as therapeutic agents¹^,²^,³^,⁴. Inclusion of cis-acting regulatory elements in the transgene expression cassette enabl....