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Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation.

Dory D, Le Moigne V, Cariolet R, Béven V, Keranflec'h A, Jestin A - J Immunol Res (2015)

Bottom Line: Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy.This is even more difficult for large and growing animals.Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

View Article: PubMed Central - PubMed

Affiliation: French Agency for Food, Environmental and Occupational Health Safety (ANSES), Viral Genetics and Biosafety Unit, 22440 Ploufragan, France.

ABSTRACT
DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

No MeSH data available.


Related in: MedlinePlus

Identification of the injection point. (a) Four dots were tattooed with Indian ink on the skin of the left biceps femoris muscle 2 to 3 weeks before the injection. (b) The injection site of the plasmids was located at the intersection of the two lines passing through these dots. These two lines were drawn on the skin just before the injection.
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Related In: Results  -  Collection


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fig1: Identification of the injection point. (a) Four dots were tattooed with Indian ink on the skin of the left biceps femoris muscle 2 to 3 weeks before the injection. (b) The injection site of the plasmids was located at the intersection of the two lines passing through these dots. These two lines were drawn on the skin just before the injection.

Mentions: The experimental protocol was approved by the ethic committee for animal experimentation of ANSES/National Veterinary School of Alfort/University of Paris-Est Créteil (France) (Notice number 10/04/13-05). Pigs were housed and treated in accordance with the requirements of the local veterinary authority. Four groups of four specific pathogen-free eight-week old pigs were used. The injection site of the plasmids was identified through four dots tattooed with Indian ink on the skin of the left biceps femoris muscle, the injection site being located at the intersection of the two lines passing through these dots (Figure 1). All pigs were anesthetized with an auricular intravenous injection of thiopental (1 g/50 kg body weight). The first and second group were injected with 2.5 × 1014 copies of PrV-gB-pcDNA3 prepared in 600 μL PBS. The third one received 2.5 × 1014 copies of pcDNA3 and the last group was injected with PBS. 0.45 mm × 12 mm needles were used. Eighty seconds later [18], electroporation which consists of 5 pulses of 150 V and 20 ms with a 200 ms interval between each pulse [7] was applied through stainless-steel electrodes (0.2 mm wires, 1 cm long, and 10 mm apart) introduced on either side of the injection point of pigs of groups 2 to 4. The electric current was applied with a BTX ECM 830 pulse generator (Harvard Apparatus, Holliston, MA, USA). Pigs were observed daily. Body temperature and body weight were measured daily and weekly, respectively. Pigs were sacrificed six weeks after injection. The muscle injection site identified through the tattooed dots was sampled using a disposable 2 cm long and 0.8 cm diameter biopsy punch (Figure 2), frozen in liquid nitrogen, and stored at −80°C until DNA extraction.


Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation.

Dory D, Le Moigne V, Cariolet R, Béven V, Keranflec'h A, Jestin A - J Immunol Res (2015)

Identification of the injection point. (a) Four dots were tattooed with Indian ink on the skin of the left biceps femoris muscle 2 to 3 weeks before the injection. (b) The injection site of the plasmids was located at the intersection of the two lines passing through these dots. These two lines were drawn on the skin just before the injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4561992&req=5

fig1: Identification of the injection point. (a) Four dots were tattooed with Indian ink on the skin of the left biceps femoris muscle 2 to 3 weeks before the injection. (b) The injection site of the plasmids was located at the intersection of the two lines passing through these dots. These two lines were drawn on the skin just before the injection.
Mentions: The experimental protocol was approved by the ethic committee for animal experimentation of ANSES/National Veterinary School of Alfort/University of Paris-Est Créteil (France) (Notice number 10/04/13-05). Pigs were housed and treated in accordance with the requirements of the local veterinary authority. Four groups of four specific pathogen-free eight-week old pigs were used. The injection site of the plasmids was identified through four dots tattooed with Indian ink on the skin of the left biceps femoris muscle, the injection site being located at the intersection of the two lines passing through these dots (Figure 1). All pigs were anesthetized with an auricular intravenous injection of thiopental (1 g/50 kg body weight). The first and second group were injected with 2.5 × 1014 copies of PrV-gB-pcDNA3 prepared in 600 μL PBS. The third one received 2.5 × 1014 copies of pcDNA3 and the last group was injected with PBS. 0.45 mm × 12 mm needles were used. Eighty seconds later [18], electroporation which consists of 5 pulses of 150 V and 20 ms with a 200 ms interval between each pulse [7] was applied through stainless-steel electrodes (0.2 mm wires, 1 cm long, and 10 mm apart) introduced on either side of the injection point of pigs of groups 2 to 4. The electric current was applied with a BTX ECM 830 pulse generator (Harvard Apparatus, Holliston, MA, USA). Pigs were observed daily. Body temperature and body weight were measured daily and weekly, respectively. Pigs were sacrificed six weeks after injection. The muscle injection site identified through the tattooed dots was sampled using a disposable 2 cm long and 0.8 cm diameter biopsy punch (Figure 2), frozen in liquid nitrogen, and stored at −80°C until DNA extraction.

Bottom Line: Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy.This is even more difficult for large and growing animals.Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

View Article: PubMed Central - PubMed

Affiliation: French Agency for Food, Environmental and Occupational Health Safety (ANSES), Viral Genetics and Biosafety Unit, 22440 Ploufragan, France.

ABSTRACT
DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

No MeSH data available.


Related in: MedlinePlus