Name: in vivo gene transfer to the rabbit common carotid artery endothelium
Uploaded: 2018-05-06T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

We thank AdVec, Inc. for permission to use HDAd reagents, Julia Feyk for administrative assistance, and the Department of Comparative Medicine veterinary services for surgical advice and support. This work was supported by HL114541 and the John L. Locke, Jr. Charitable Trust.

All methods described here were approved by the University of Washington Office of Animal Welfare and associated Institutional Animal Care and Use Committee (IACUC), and were completed in accordance and compliance with all relevant regulatory and institutional guidelines.
Note: Gene transfer to rabbit common carotid arteries is performed on rabbits by a surgeon with the aid of an anesthesiologist or assistant.
1. Gene transfer to rabbit common carotid arteries: Pre-operation
<ol>
	<li>Anesthetize the rabb...

Certain aspects of surgical technique merit particular attention. Full exposure and mobilization of the common carotid artery via careful dissection will facilitate gene transfer and arteriotomy repair. However, during the dissection, direct manipulation of the carotid artery should be minimized&#160;to prevent vasospasm. In addition, any bleeding adjacent to the artery should be stopped by applying light pressure with gauze and extravasated blood should be cleaned up immediately by rinsing the area with normal saline. I...

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 enables assessment of the activity of these elements in arterial endothelium in vivo5,6. Knowledge of the activity of specific cis-acting regulatory elements can be used to design more-active expression cassettes and to probe mechanisms of gene regulation in large artery endothelium in vivo7.
Rabbits are a valuable model for various aspects of human vascular physiology and disease. Rabbits share many vascular features with humans. For example, baseline hematological values, hemostatic regulation, and vascular longitudinal tension are similar between rabbits and humans8. Rabbit models of vascular diseases replicate key features of many human diseases including: aneurysms (similar geometric and flow characteristics)9, vasospasm (similar response to endovascular treatment)10,11, and atherosclerosis (intimal plaques with similar features including a core rich in lipid, macrophages, and smooth muscle cells in a fibrous cap)12,13. Accordingly, rabbit models have been developed for many vascular diseases such as thrombosis, vasospasm, aneurysm, diabetes, vascular graft stenosis, and atherosclerosis8,13,14,15,16.
For researchers choosing among animal models of vascular physiology and disease, the rabbit has several advantages. Compared to rodents, the larger vessels of rabbits allow easier surgical manipulation, use of endovascular devices, and a larger amount of tissue for quantitative measurements. Rabbits are much closer phylogenetically to primates than are rodents17, and the greater genetic diversity of outbred rabbits better approximates the genetic variability of humans. Genetic diversity is particularly important for preclinical studies, which-by their nature-aim to develop therapies that can be applied to the genetically diverse human population. As with many if not all other model species, rabbit genes are easily cloned or synthesized because the rabbit genome has been sequenced with high coverage (7.48x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway?db=oryCun2]. Compared to other large animal models (such as dogs, pigs, or sheep), rabbits are relatively inexpensive to purchase and house and they are easier to breed and handle. Specific vascular disease models in rabbits each have their own advantages and shortcomings as models of human disease that are beyond the scope of this manuscript8,12,18. An investigator should review these advantages and shortcomings to determine if the rabbit is the best model for answering a specific experimental question.
Introduction of deoxyribonucleic acid (DNA) regulatory sequences into endothelial cells in vivo enables investigation of the activity of these sequences in a complex physiologic environment. In vitro studies in transfected endothelial cells can be useful for the initial assessment of DNA regulatory sequences; however, expression levels in tissue culture models are sometimes not reproduced when the studies are repeated in vivo5,19,20. In vitro systems can also be useful for exploring basic pathways of protein signaling and endothelial physiology as well as communication between cultured vascular cells; however, more-complex pathways or regulatory networks that are influenced by complex populations of neighboring vascular cells or the immune system are best studied in an in vivo system6,20. The method described herein provides a platform for exploring regulation of transgene expression in the endothelium within the context of an intact vessel, with or without disease. The in vivo system also permits investigation of physiological and pathological cellular crosstalk and identification of contributions of the immune system to regulation of gene expression6.
Germ-line transgenesis (especially in mice) is an alternative approach for directing transgene expression to endothelial cells. This approach can provide life-long transgene expression, with endothelial targeting mediated by specific promoter or regulatory regions21,22. However, the generation of transgenic mice is time-consuming and expensive, several transgenic lines must be often tested to ensure targeting of the transgene to the desired cell type and achievement of adequate transgene expression levels, and experimental results in murine systems can be strain-dependent. Murine transgenic models with endothelial-targeted transgenes have many advantages: there is no need to perform surgery on every experimental animal in order to achieve transgenesis, experimental mice can be bred with numerous other available transgenic mice in order to test genetic and phenotypic interactions, and there is a wide selection of antibodies that react with murine proteins, facilitating characterization of phenotypes. However, targeting of transgenes to the endothelium via the germ line typically results in transgene expression throughout the vasculature,22 making it difficult to determine the site at which the transgene product is acting. This is especially true when the transgene product is secreted, because a transgene product secreted by endothelial cells throughout the vasculature could have biological activity at any number of sites within an animal. Although the method described in this manuscript requires technical expertise and specialized facilities, it can be less time consuming and less expensive than developing an endothelial-specific transgenic mouse line. It allows for the assessment of the function of a protein selectively in endothelial cells of a segment of large artery, and it permits use of the contralateral common carotid as a paired control (eliminating systemic factors that can vary among experimental animals-for example, blood pressure or cholesterol levels-as uncontrolled variables).
Gene therapy is a promising approach for the treatment of vascular diseases, particularly chronic diseases, because a single application can provide sustained or possibly life-long expression of a therapeutic gene23. The therapeutic promise of gene therapy has been explored in animal models of somatic gene transfer, often targeting the liver24,25, which is a relatively easy target because many blood-borne viral vectors are hepatotropic. However, to have an effect on vascular disease, gene therapy targeted to the liver must achieve systemic overexpression of proteins. This typically requires large doses of vector, which can be toxic or even fatal26. Moreover, increased systemic levels of a protein raise the risk of off-target side effects, which could complicate or even obscure interpretation of experimental results. Local gene therapy targeting vascular endothelium as described in this manuscript could avoid systemic side effects because the infused vector is not widely disseminated beyond the transduced arterial segment, and local vascular effects can be achieved without changes in systemic plasma levels of protein.27 In addition, a far lower amount of vector is needed to transduce an arterial segment than is needed to achieve robust hepatic transduction. Transgene expression from the liver has been reported to decline over time, probably due to cell turnover, requiring repeated dosing if high-level transgene expression is to be maintained.28 In contrast, the low turnover rate of the endothelium provides stable expression for at least 48 weeks in chow-fed rabbits and for at least 24 weeks in atherosclerotic lesions of cholesterol-fed rabbits.1,27
To determine if this method of gene transfer to rabbit common carotid endothelium is appropriate, the advantages and disadvantages (Table 1) should be considered in the context of the specific research goals. Advantages of this method include: outbred rabbits are better representative of human genetic diversity than are inbred mice (important for preclinical work); rabbits provide larger vessels for easier manipulation and more tissue for analysis; the method can achieve endothelium-targeted transgene expression far more quickly than does germ-line endothelial targeting in transgenic mice; vector dose can be easily adjusted to model variable levels of transgene expression; processes specific to large-artery endothelium can be investigated; and local vascular transgenesis allows the opposite carotid in the same animal to be used as a control, eliminating systemic factors as uncontrolled variables. Disadvantages include: special facilities and expertise are required; fewer genetically modified backgrounds on which to experiment are available in rabbits than in mice; and there is a less extensive selection of antibodies to rabbit versus mouse proteins (for immunodetection of transgene protein and other antigens that may be important in interpreting experimental results).

<table border="0" cellpadding="0" cellspacing="0" style="width:888px;" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Disposables</td>
			<td style="width:181px;"></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">3mL syringe with 24G needle</td>
			<td>Becton Dickinson</td>
			<td>309571</td>
			<td style="width:199px;">2x for gene transfer surgery; 3x for harvest surgery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">1mL syringe with 27G needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td style="width:199px;">6x for gene transfer surgery; 1x for harvest surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">20mL syringe, luer lock</td>
			<td>Nipro Medical Corp</td>
			<td>JD+20L</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheters, 24G x 3/4&#34;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">19G needle</td>
			<td>Becton Dickinson</td>
			<td>305187</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">21G needle</td>
			<td>Becton Dickinson</td>
			<td>305165</td>
			<td style="width:199px;">For 20 mL syringe of saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Gauze 4&#34; x 4&#34;</td>
			<td>Dynarex</td>
			<td style="width:149px;">3242</td>
			<td style="width:199px;">~10-15 per surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-244</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-182</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8648P</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>398A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alcohol swabs</td>
			<td>Covidien Ltd.</td>
			<td>6818</td>
			<td style="width:199px;">For placement of I.V. line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheter plug</td>
			<td>Vetoquinol</td>
			<td>411498</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Ketamine HCl, 100 mg/mL</td>
			<td>Vedco Inc.</td>
			<td>05098916106</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Xylazine, 100 mg/mL</td>
			<td>Akorn Inc.</td>
			<td>4821</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Lidocaine HCl, 2%</td>
			<td>Pfizer</td>
			<td>00409427702</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Bupivacaine HCl, 0.5%</td>
			<td>Pfizer</td>
			<td>00409161050</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Beuthanasia D-Special</td>
			<td>Intervet Inc.</td>
			<td>NDC 00061047305</td>
			<td style="width:199px;">Harvest surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Buprenorphine HCl, 0.3 mg/mL&#160;</td>
			<td>Patterson Veterinary</td>
			<td>12496075705</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Saline IV bag, 0.9% sodium chloride</td>
			<td>Baxter</td>
			<td>2B1309</td>
			<td style="width:199px;">2x for gene transfer surgery; can use vial of sterile saline in place of one</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Heparin&#160; (5000 U/mL)</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-047-10</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Fentanyl patch, 25 mcg/hr&#160;</td>
			<td>Apotex Corp.</td>
			<td>NDC 60505-7006-2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:359px;">Gene transfer vector</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;">Dilute 350 &#181;L per artery; 2 x 1011 vp/mL for adenovirus; gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Instruments</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Metzenbaum needle holder 7&#34; straight</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7900</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Operating scissors 6.5&#34; straight blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6828</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Needle holder /w suture scissors</td>
			<td>Miltex</td>
			<td style="width:149px;">8-14-IMC</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo needle holder, 5.75&#34; straight with lock</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6412</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Stevens scissors 4.25&#34; curved blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5943</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alm retractor 4&#34; 4X4 5mm blunt prongs</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6514</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Backhaus towel clamp 3.5&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">4x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Micro clip setting forceps 4.75&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6496</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Micro vascular clips, 11 mm</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">2x for gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surg-I-Loop</td>
			<td>Scanlan International</td>
			<td style="width:149px;">1001-81M</td>
			<td style="width:199px;">5 cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Bonaccolto forceps, 4&#8221; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5210</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Dumont #3 forceps Inox tip size .17 X .10mm</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5042</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Graefe forceps, 4&#8221; (10 cm) long serrated straight, 0.8mm tip</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5280</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; straight, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7110</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; curved, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7111</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Jacobson mosquito forceps 5&#34; curved extra delicate, 0.9 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7117</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Kantrowitz forceps, 7.25&#34; 90 degree delicate, 1.7 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7305</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Tissue forceps 5&#34;, 1X2 teeth, 2 mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-8162</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11092-12</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Adson forceps, 12 cm, serrated, straight</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11006-12</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery handpiece and electrode</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">HPAC-1; R-F11</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Suite Equipment</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Circulating warm water blanket and pump</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Forced air warming unit</td>
			<td>3M</td>
			<td style="width:149px;">Bair Hugger Model 505</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">IV infusion pump</td>
			<td>Heska</td>
			<td style="width:149px;">Vet IV 2.2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane vaporizer and scavenger</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary multi-parameter monitor</td>
			<td>Surgivet</td>
			<td style="width:149px;">Surgivet Advisor</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery unit</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">MV-9</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical microscope</td>
			<td style="width:181px;">D.F. Vasconcellos</td>
			<td style="width:149px;">M900</td>
			<td style="width:199px;">Needs ~16x magnification</td>
		</tr>
	</tbody>
</table>

in vivo gene transfer to the rabbit common carotid artery endothelium

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.

To implement this method with confidence, preliminary experiments are necessary to establish that the operator achieves efficient and reproducible gene transfer, with transgene expression primarily in luminal endothelial cells. In our experience, this is most easily assessed using a vector that expresses &#946;-galactosidase. 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal) staining of common carotid segments removed 3 days after vector infusion, as well as measurement of &#9...

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In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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 ...

The overall goal of this surgical procedure is to introduce a transgene into the endothelial cells of rabbit carotid arteries. This method helps answer questions in vascular biology and gene therapy about the roles of arterial wall transgene products and the roles of DNA-regulatory sequences in controlling transgene expression. This technique allows the efficient expression of transgenes within a focal segment of the vascular endothelium, allowing the site-specific study of the biological roles of endothelial-expressed genes.Generally, individuals new to this method will struggle as new investigators often puncture back wall of the artery and do not tie off all of the small side branches. After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and shave both ears and the left-rear middle toe. Insert a 24 gauge IV catheter into the left ear vein and apply a fentanyl patch to the right ear.Shave the anterior neck from the sternal notch to the edge of the mandible. Place the rabbit in the supine position on the operating table with a rolled neck support towel just below the animal's head. Gently extend the rabbit's neck until it is straight and approximately horizontal.To begin the surgery, use electrocautery to cut the skin approximately seven to nine centimeters along the midline. Clamp the tissue open with towel clamps and make a short lateral cut through the fascia with the electrocautery. Insert large scissors into the cut to bluntly dissect the fascia along the midline and cut through the dissected fascia with the electrocautery device.Beginning on the right side, use small dissecting scissors to dissect between the V-shaped sternocephalic muscles and the sternohyoid muscle over the trachea to expose the common carotid arteries. Dissect the right common carotid artery from the surrounding tissues from the base of the neck caudally to the crossing of the pharyngeal nerve cranially and use surgical silicon loops to aid in the retraction of the artery. Ligate any branches coming off of each side of the common carotid artery with 5-0 silk sutures.Then cut each branch distally one to two millimeters away from the sutures to free a four to five centimeter segment of the carotid. Next, move a surgical microscope into position and drape a sterile towel over the microscope to allow manipulation of the microscope without contaminating the sterile field. Place two silk ties, each with a loose overhand knot, around the midportion of the mobilized carotid segment.Place a vascular clip at the cranial end of the isolated artery segment to allow the artery to fill, then place a vascular clip at the caudal end of the artery. Using a 19 gauge needle bent just above the bevel at an approximately 80-degree angle, puncture the artery just cranial to the caudal vascular clip, taking care not to puncture the back or side walls of the vessel. Advance the needle tip into the lumen, withdrawing the tip twice to make sure the arteriotomy completely traverses the artery wall before carefully withdrawing the needle completely.Next, bend an IV catheter about four millimeters from the tip to an approximately 75-degree angle and loosely attach the IV catheter to a syringe filled with sterile tissue culture medium. Insert the catheter into the artery up to the bend point and fill the artery with 250 microliters of medium, then gently press with a gloved finger along the artery from just caudal to the cranial vascular clip to the arteriotomy to flush out the medium. After a second wash as just demonstrated, tighten the two silk sutures around the catheter tip to seal the lumen and attach a syringe containing the virus solution to the catheter.Gently depress the syringe plunger to infuse 250 microliters of the virus until the artery is distended to its physiological caliber and gently lay the syringe on a nest of gauze for support. Place a 7-0 polypropylene suture in the common carotid adventitia just caudal to the cranial vascular clip to mark the cranial boundary of gene transduction. After 20 minutes, replace the virus-containing syringe with an empty syringe and gently aspirate the virus-containing solution until the vessel collapses.Then remove the syringe, silk ties, and catheter. Using 7-0 polypropylene suture, make a loose X-pattern across the arteriotomy. Very briefly release the cranial vascular clip to flush the artery of any air or residual virus and tighten the suture.Release the cranial and caudal vascular clips using light pressure and gauze to stop any bleeding and close the midline fascia with a continuous suture, then close the skin with an intradermal pattern with buried knots at both ends. To harvest the transduced carotid arteries at the appropriate experimental endpoint, use 3-0 silk suture to ligate the common carotid artery cranially and caudally to the segment infused with the vector. The 7-0 suture placed in the adventitia marks the cranial end and the arteriotomy marks the caudal end.Excise the carotid segment between the ligations and flush the lumen with saline. Then trim away any excess adventitia and cut the transduced carotid segment into smaller pieces for the appropriate experimental endpoint analyses. En face images of the lumenal surfaces of rabbit common carotid arteries infused with adenovirus expressing beta-galactosidase demonstrate a robust endothelial staining with X-gal.Axial images of the lumenal surfaces of intact carotid rings reveal X-gal staining primarily on the lumenal surface. X-gal staining is also observed primarily in the endothelial cells of axially-opened vessels, although a small amount of staining is found in the adventitial layer. Analysis of RNA extracted from the vessel segments reveals substantial over-expression of transgenes relative to their endogenous levels, with considerable intra-and inter-artery variability in transduction efficiency and transgene expression observed among arteries transduced by experienced operators.Once mastered, the rabbit common carotid artery gene transfer surgical procedure can be completed in less than two hours. Following this procedure, RT-PCR, immunohistochemistry, Western blotting, or other methods can be performed to answer questions about the role of DNA-regulatory sequences in altering transgene expression and the role of transgene products within the artery wall. After its development, this technique paved the way for the study of gene therapy in treatment of vascular diseases such as arterial atherosclerosis and of vein graft disease.Don't forget proper bio-safety precautions should always be taken when working with viral vectors and sharp objects.

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