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For the exact evaluation, whether the plasmid is integrated into genomic DNA or presented in extrachromosomal state, a precise gel purification method would be necessary [ 4 , 5 , 12 , 13 ].
Furthermore, plasmid integration into genomic DNA is a very rare event, usually lower than the level of spontaneous mutation [ 4 ].
Wang et al. According to such calculations, plasmid integration into genomic DNA in our experiments would be mostly at the level below quantification limit or even undetectable.
Cationic liposomes are mostly used as carries for intravenous systemic delivery, but novel lipid combinations might be suitable for i.
When we compared the cationic liposomes with the standard method of i. Generally, our data demonstrated a slower clearance of pDNA from the injection site of pDNA:CDAN-DOPE group within the period of day 1 and day 28 in comparison to both, the naked and electroporated groups Fig.
This data would support the consideration that pDNA in liposomal complex is more protected against the attack of nuclease. With regards to the observation of Hartikka et al[ 43 ], who noticed that another cationic lipid formulation — Vaxfectin did not appear to increase transfection, we can suppose that high plasmid levels are located extracellulary.
Using fluorescently labelled liposomes, histological analysis revealed that liposomal complexes were mostly distributed along the injection lane, forming a depot within muscle tissues even after 28 days Fig.
In order to facilitate further plasmid detection and potentially minimise a condemnation of whole consumable parts, coccygeus muscle was chosen as a suitable site for immunization.
It is important to note that this small muscle, located closely to the root of the tail, is easy to reach and remove after slaughter.
Having 10 animals available in experimental herd, we tested i. Unfortunately, we had not suitable electrodes for the electroporation of larger animals at the time of the experiments.
Instead of electroporation we applied pDNA vaccine in combination with liposomal adjuvant BnorAbu-MDP, which was proved to be effective in guinea pigs immunized by the same pDNA vaccine unpublished results.
Altogether, the performed QRTPCR assay revealed that pDNA persisted in ultra-low level at the injection site even days after the second administration of pDNA.
The highest amount of pDNA was detected in the group vaccinated by pDNA:cationic liposome complexes. These data are in good accordance with the results obtained in mice.
The values of residual pDNA in the group injected by naked pDNA were mainly non-quantifiable. Combination of naked pDNA with the liposomal adjuvant BnorAbu-MDP resulted in levels of residual pDNA close to quantification limit.
It is important to emphasize that no plasmid was detected in distant muscle tissue, in draining lymph node or in the opposite muscle directly connected with these lymph nodes.
The tissues located contralaterally to the injection sites could also be considered as negative controls for each vaccinated animal.
Quantitative real-time PCR QRTPCR assay was developed to assess a residual pDNA vaccine pVAX-Hsp60 TM in mice and beef cattle.
In beef cattle, ultra low residual level of pDNA vaccine was found only at the site of injection. Residual plasmid in native state will hardly be found at measurable level following further meat.
PO carried out development of QRTPCR, participated in quantification of pDNA, and participated in preparation of the manuscript. VK participated in preparation of cationic liposomes, carried out the histology experiments and electroporation.
MR designed and prepared the plasmid for vaccination and participated in preparation of the manuscript. ADM designed and synthesised cationic lipids.
ML designed and synthesised muramylglycopeptide adjuvans. JT conceived of the study, participated in its design and coordination, prepared and characterised liposomes, performed immunisation experiments and drafted the manuscript.
All authors read and approved the final manuscript. This work was supported by grant NAZV QF , the Ministry of Agriculture of the Czech Republic grant No.
MZE and MSM We also thank IC-Vec Ltd, UK for support. National Center for Biotechnology Information , U. National Library of Medicine Rockville Pike , Bethesda MD , USA.
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Journal List Genet Vaccines Ther v. Genet Vaccines Ther. Published online Sep 2. PMCID: PMC Andrew D Miller 3 Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College London, London, SW7 2AZ, UK Find articles by Andrew D Miller.
Miroslav Ledvina 4 The Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic Find articles by Miroslav Ledvina.
Author information Article notes Copyright and License information Disclaimer. Corresponding author.
Received May 15; Accepted Sep 2. This article has been cited by other articles in PMC. Abstract Background Application of plasmid DNA for immunization of food-producing animals established new standards of food safety.
Methods A quantitative real-time PCR QRTPCR methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX pVAX-Hsp60 TM in mice and beef cattle.
Results Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent naked pDNA, electroporation, cationic liposome complexes and mouse age-dependent clearance form injection site but pDNAX was still detectable even after days.
Conclusion Quantitative real-time PCR QRTPCR assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM in mice and beef cattle.
Background DNA-based vaccines represent a new and rapidly progressing area in vaccinology. Materials and methods Plasmids The plasmid pDNAX pVAX-Hsp60 TM , encoding the heat shock protein 60 Hsp60 from Trichophyton mentagrophytes [ 24 ] and the plasmid pLacZ pcDNA3.
DNA extraction from tissue sample The isolation of genomic DNA gDNA from the samples of tissue taken from mice or beef cattle was performed by modification of guanidine thiocyanate GuSCN lysis method followed by binding of DNA to SiO 2 [ 26 ].
QRTPCR analysis The Genecompare software Applied-Maths, Belgium was used to design primers amplifying a sequence stretch that contains plasmid specific promoter sequence CMV as well as sequence from hsp60 gene, generating bp specific product.
Precautions to prevent contamination All the manipulations with stock plasmid, tissue sampling, QRTPCR set up and template addition were done in separated working areas [ 27 ].
Results Validation of QRTPCR method Persistence of pDNAX was determined by a QRTPCR methodology designed to specifically recognize the stretch of promoter-insert from the pDNAX plasmid.
Open in a separate window. Figure 1. Figure 2. Experiment II Identical experiment was performed with 5-week-old mice to evaluate a possible relationship between the animal age and the rate of clearance of pDNAX from the site of injection.
Figure 3. Figure 4. Table 1 Effect of various pDNAX formulations on its persistence in beef cattle after i. General safety After the injection of pDNAX or pLacZ , both mice and cows from all the tested groups survived throughout the duration of the experiments and neither any apparent pathological changes at the site of injection nor loss of body weight were observed indicating that pDNAX pVAX-Hsp60 TM vaccine and its formulations as a complex with cationic liposomes or liposomal adjuvant BnorAbu-MDP were well tolerated in both species.
Discussion Limited data on the examination of the effect of pDNA vaccines on food-producing animals have been reported so far and we can only extrapolate the results obtained in the model animals.
Biodistribution and persistence of pDNA in mice Model studies on rodents covering overall biodistribution and safety features are required before DNA vaccines enter human clinical trials [ 29 ].
Biodistribution and persistence of pDNA in beef cattle In order to facilitate further plasmid detection and potentially minimise a condemnation of whole consumable parts, coccygeus muscle was chosen as a suitable site for immunization.
Conclusion Quantitative real-time PCR QRTPCR assay was developed to assess a residual pDNA vaccine pVAX-Hsp60 TM in mice and beef cattle.
Competing interests The authors declare that they have no competing interests. Authors' contributions PO carried out development of QRTPCR, participated in quantification of pDNA, and participated in preparation of the manuscript.
Acknowledgements This work was supported by grant NAZV QF , the Ministry of Agriculture of the Czech Republic grant No. References Alarcon JB, Waine GW, McManus DP.
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Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization. He practices conscientiously at the swimming association and does extra training on his own For an enhanced browsing experience, get the IMDb app on your smartphone or tablet.
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