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The Transfection of Ischemic Skin Wounds with Reporter Genes Chloramphenicol Acetyltransferase and Beta Galactosidase

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Title:
The Transfection of Ischemic Skin Wounds with Reporter Genes Chloramphenicol Acetyltransferase and Beta Galactosidase
Creator:
Brinsko, Kelly
Schultz, Gregory ( Mentor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Language:
English

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serial ( sobekcm )

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University of Florida
Holding Location:
University of Florida
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All applicable rights reserved by the source institution and holding location.

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The Transfection of Ischemic Skin Wounds with Reporter
Genes Chtoramphenicot Acetyltransferase and Beta Gatactosidase

Kelly Brinsko


ABSTRACT


Chloramphenicol Acetyltransferase (CAT) and Beta Galactosidase (B-gal) were used as reporter genes to determine

if lipid:plasmid complexes could be successfully transfected into ischemic mammalian tissue. The amount of

time available for such gene therapy, the level of transfection, and the size of the transfected area were the

chief objectives of this experiment. It was found that within the most ischemic area of tissue average levels of

CAT protein increased from 0.43 � 0.05 ng immediately after surgery, day 0, to 2.06 � 0.60 ng CAT on day 6

after surgery. Day 8 registered a decrease in CAT protein to 1.61 � 0.11 ng. Furthermore, B-gal staining of

an ischemic tissue injected with a lipid:plasmid complex on day 4 after surgery displays a transfection area of 5

mm diameter. The data suggests that lipid:plasmid complexes can successfully transfect ischemic tissues even up

to eight days after surgery.



INTRODUCTION


Ischemia is a skin condition that develops where there are insufficient oxygen levels being delivered to tissues.

Often ischemia is associated with poor vascularization of the tissues, as seen in cases of venous hypertension

and diabetes. The tissue eventually hardens, turns black, and dies. When an ischemic skin condition develops,

the wound healing process is halted. Cytokines, proteases, protease inhibitors, and growth factors play intrinsic

roles in normal wound healing. Ischemia prevents these agents from either reaching the tissues or

functioning correctly.



Recently, gene therapy has been proposed for the treatment of such wounds. Neovascularization of tissues

by vascular endothelial growth factor (VEGF) would increase oxygen levels to prevent the onset of ischemia.

The common method for introducing genetic factors such as VEGF into mammalian cells involves the use of

lipid:plasmid complexes. However, it must first be determined if plasmid transfection of ischemic skin can

be achieved. The reporter genes chloramphenicol acetyltransferase (CAT) and beta galactosidase (B-gal) were

used to both quantitatively and visually determine the level of transfection. The use of CAT as a reporter gene

will demonstrate the most effective time to administer growth factor into ischemic skin. B-gal will indicate the size





of the transfection area&emdash;transfected cells will become blue after treatment. These two reporter genes

will thus lay the groundwork for research of VEGF as a therapy for ischemia.



MATERIALS AND METHODS


1. The animal model used was based on the skin flap developed by McFarlane and associates. This single

pedicle ischemic skin flap, measuring 3 cm x 11.5 cm, extends from the scapula to the iliac crest. The flap was

lifted on Sprague Dawley male rats and immediately closed using stainless steel wound clips. The flap was

marked into three equal sections: proximal at the scapula, distal at the iliac crest, and medial at the center of

the flap. (Figure 1)


Figure 1. Diagram of single pedicle ischemic skin flap. The proximal, medial, and distal segments

denoted by P, M, and D, respectively. Injections were made at the midpoints of the proximal and

distal sections.



2. CLZ plasmid delivered the beta galactosidase gene. The lipid: plasmid complex was Two different cationic

lipid: plasmid complexes were prepared. A ratio of 25 nmol of liposomes to 50 pg of reporter gene plasmid was

used. The gene expressing chloramphenicol acetyltransferase was obtained via pMP6-CAT plasmid, while

pTR-suspended in a final volume of 100 pl with PBS.



3. The lipid: plasmid complex was injected intradermally into the flap. One 100 pl aliquot of the pMP6-CAT lipid:

DNA complex was inoculated at the midpoint of both the proximal and distal segments of the flap. Five groups of

four rats each were injected on the day of surgery (day 0), 2, 4, 6, and 8 days after surgery.



4. Forty-eight hours after injection the flap was removed and separated into the proximal, medial, and

distal segments. The segments were weighed and homogenized at a ratio of 0.66 g/ml of lysis buffer (10 mM





Tris pH=7.4, 150 mM NaCI, 1% Triton-X-100), then centrifuged at 14,000xg for 10 minutes at 40C. The

supernatant from each sample was then collected and the quantity of CAT protein was measured using a

specific enzyme-linked immunosorbant assay (ELISA). The reading was considered as the total amount of

CAT activity present in the particular segment of the flap.



5. The pTR-CLZ lipid:plasmid complex was injected at the time of surgery in the center of the distal segment.

Tissue was collected 48 hours later, fixed in 4% paraformaldehyde for two hours at 40C, and assessed with a B-

gal colorimetric assay. The area of gene transfer turned blue for elementary observation.



RESULTS


Using a standard curve set by the ELISA, the rate of transfection of CAT was determined (Figure 2). Within the

first 24 hours after surgery, the transfection efficiency of all segments of the flap is approximately equal to 0.4

ng. However, as evidenced by the graph, the transfection of CAT protein in the distal area of the flap begins

to increase as early as 2 days after surgery. The transfection rate peaks at day 6, where 2.06 � 0.60 ng CAT

were detected. This level decreases to 1.61 � 0.11 ng on day 8. By statistical analysis, the values on days 6 and

8 were significantly higher (p<0.001 by Tukey's HSD post-ANOVA test) than levels in the proximal and

distal segments. It is also noteworthy to highlight the fact that CAT protein levels in the proximal and distal

sections of the flap were never greater than 1.00 ng.


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Day 0 Day 2 Day 4 Day 6 Day 8
Day of Plamsid Injection After Elevation of Flap


Figure 2. Graph depicting the amount of CAT protein present in the proximal, medial, and distal

sections of the flap at days 0, 2, 4, 6, and 8. Days 6 and 8 are statistically significant.



Examination of the flaps injected intradermally with the B-gal lipid:plasmid complex reveal a blue spot with

a diameter of approximately 5 mm (Figure 3). The circular area indicates that the scope of a single injection is

of workable size surrounding the injection site.

























Figure 3. Distal portion of flap showing localization of transfection as it appears above (left) and

below (right) the derma.




DISCUSSION


The implications of this experiment are extensive. It has been successfully shown that gene therapy is possible

on chronic wounds with little or no vascularization. This is an important point since most gene therapy today relies

on blood vessels to deliver the gene product. With the use of intradermal injections of lipid:DNA

complexes, therapeutic genes can be delivered directly to the desired area. In addition, the therapy can

be administered even 8 days after surgery. This is very important in a medical setting, where ischemia is

not detectable until about 2-4 days after surgery. The patient can be successfully treated after the wound

becomes ischemic.



The B-gal experiment produced an area of transfection around the injection site of approximately 5 mm

diameter. This result also applies to practical medical uses in that the number of injection sites, as well as

their placement in the wound, can be easily determined.



Now that it has been successfully shown that gene therapy is possible in ischemic skin, growth factors (such as

VEGF) and other agents involved in wound healing can be extensively explored.






REFERENCES


1. Robson MC. The Role of Growth Factors in the Healing of Chronic Wounds. Wound Repair and Regeneration.

1997; 5:12-17.

2. Mast BA, Schultz GS. Interaction of cytokines, growth factors,a nd preoteases in acute and chronic wounds.

Wound Repair and Regeneration. 1996; 4:411-420.





3. Tarnuzzer RW, Schultz GS. Biochemical Analysis of Acute and Chronic Wound Environments. Wound Repair

and Regeneration. 1996; 20:670-674.

4. Nail AV, Brownlee RE, Colvin CP, Schultz G, Fein D, Cassisi NJ et al. Transforming Growth Factor bl Improves

Wound Healing and Random Flap Survival in Normal and Irradiated Rats. Archives of Otolaryngology Head and

Neck Surgery. 1996; 122:171-177.

5. McFarlane RM, DeYoung G, Henry RA. The Design of a Pedical Flap in the Rat to Study Necrosis and Its

Prevention. Plastic and Reconstructive Surgery. 1965; 35:177-182.


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