We would like to thank all members of the Kong Lab and Dr. Yan Deng at Microscopy Core, Office of Collaborative Science, NYUMC for their assistance and technical support. This work was supported by a pilot grant from the New York University Center for AIDS Research (CFAR, NIH grant AI027742).

1. Plasmid DNA Purification
<ol>
 <li>Transform the eukaryotic plasmid DNA encoding EGFP (pCX-EGFP) into DH5&alpha; E.coli competent cells. The empty pCX vector may also be used as a negative control. </li>
 <li>Culture and harvest the DH5&alpha; E.coli cells and purify the pDNA according to the Qiagen EndoFree Plasmid Purification Handbook. </li>
 <li>Filter pDNA solution through a 0.22 &mu;m PVDF sterile filter, and store it at -20 &deg;C until use. </li>
</ol>
2. Tattoo...

<ol>
	<li>Bins, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+potent+DNA+vaccination+strategy+defined+by+in+vivo+monitoring+of+antigen+expression.">A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression.</a> Nat Med. 11, 899-904 (2005).</li><li>Pokorna, D., Rubio, I., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-vaccination+via+tattooing+induces+stronger+humoral+and+cellular+immune+responses+than+intramuscular+delivery+supported+by+molecular+adjuvants.">DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants.</a> Genet. Vaccines Ther. 6, 4 (2008).</li><li>van den Berg, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intradermal+vaccination+by+DNA+tattooing+in+human+skin.">Optimization of intradermal vaccination by DNA tattooing in human skin.</a> Hum. Gene Ther. 20, 181-189 (2009).</li><li>Liu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines:+a+review.">DNA vaccines: a review.</a> J. Intern. Med. 253, 402-410 (2003).</li><li>Koide, Y., Nagata, T., Yoshida, A., Uchijima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines.">DNA vaccines.</a> Jpn. J. Pharmacol. 83, 167-174 (2000).</li><li>Peachman, K. K., Rao, M., Alving, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+DNA+through+the+skin.">Immunization with DNA through the skin.</a> Methods. 31, 232-242 (2003).</li><li>Verstrepen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+HIV-1+specific+T-cell+responses+by+short-interval+DNA+tattooing+as+compared+to+intramuscular+immunization+in+non-human+primates.">Improved HIV-1 specific T-cell responses by short-interval DNA tattooing as compared to intramuscular immunization in non-human primates.</a> Vaccine. 26, 3346-3351 (2008).</li><li>Potthoff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+and+efficacy+of+intradermal+tattoo+immunization+with+adenoviral+vector+vaccines.">Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines.</a> Vaccine. 27, 2768-2774 (2009).</li><li>Quaak, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+tattoo+vaccination:+effect+on+plasmid+purity+and+transfection+efficiency+of+different+topoisoforms.">DNA tattoo vaccination: effect on plasmid purity and transfection efficiency of different topoisoforms.</a> J. Control Release. 139, 153-159 (2009).</li><li>Pokorna, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+with+human+papillomavirus+type+16-derived+peptides+using+a+tattoo+device.">Vaccination with human papillomavirus type 16-derived peptides using a tattoo device.</a> Vaccine. 27, 3519-3529 (2009).</li></ol>

DNA vaccination is considered safer than traditional vaccination strategies as it does not require manipulation of, or expose the vaccines to, live or attenuated pathogens4. However, the result of DNA vaccination depends heavily on the delivery route. Skin is abundant in antigen-presenting cells, such as Langerhans Cells and dendritic cells1, and thus an ideal site for immunization in terms of immunogenicity and ease of access5,6. As a result, intradermal vaccination strategy is one of th...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>pCX-EGFP plasmid DNA </td>
 <td>Clontech</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>STEALTH Rotary Tattoo System </td>
 <td>Worldwide Tattoo Supply</td>
 <td>STEALTH-L</td>
 <td></td>
 </tr>
 <tr>
 <td>Tattoo needles </td>
 <td>Worldwide Tattoo Supply</td>
 <td>1207RSB</td>
 <td></td>
 </tr>
 <tr>
 <td>EndoFree Plasmid Maxi Kit </td>
 <td>Qiagen</td>
 <td>12362</td>
 <td></td>
 </tr>
 <tr>
 <td>0.22 &mu;m PVDF sterile filter </td>
 <td>Millipore</td>
 <td>SLGV013SL</td>
 <td></td>
 </tr>
 <tr>
 <td>electrical hair trimmer</td>
 <td>Commercially available </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>disposable safety razors</td>
 <td>Commercially available </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Silver Sulfadiazine Cream</td>
 <td>Watson</td>
 <td>NDC 0591-0810-55</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine HCL</td>
 <td></td>
 <td>NDC 0856-2012-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Zylazine Sterile Solution</td>
 <td></td>
 <td>NADA 139-236</td>
 <td></td>
 </tr>
 </tbody>
</table>

skin tattooing as a novel approach for dna vaccine delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

The expression of EGFP with an excitation peak at 488 nm and emission peak at 509 nm can be observed in mouse skin cells. From a 1.875 &mu;g dose of DNA, containing approximately 3&times;1017copies of the plasmid, we typically observed 10-20 EGFP signals in the 1 cm2 tattooed area. This relatively low number of transfected cells is consistent with the results of a previous study3. The EGFP expression (Figure 1) provides the evidence that EGFP plasmid was delivered into th...

Watch this Scientific Journal Video about Skin Tattooing As A Novel Approach For DNA Vaccine Delivery at JoVE.com

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

skin-tattooing-as-a-novel-approach-for-dna-vaccine-delivery

Research

JoVE Journal

Bioengineering

15.8K 次观看.  New York University School of Medicine. 皮肤纹身是一个强大的和安全的方式来传递DNA疫苗皮内。在这里，一个DNA质粒EGFP交付纹身实验室小鼠的皮肤，和EGFP在皮肤细胞中的表达，然后通过激光共聚焦显微镜检查。