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dc.contributor.author
Forcato, Diego Oscar
dc.contributor.author
Olmos Nicotra, Maria Florencia
dc.contributor.author
Ortega, Nicolás Matías
dc.contributor.author
Alessio, Ana Paula
dc.contributor.author
Fili, Alejandro
dc.contributor.author
Rodriguez, Natalia Evelin
dc.contributor.author
Bosch, Pablo
dc.date.available
2017-08-22T17:58:07Z
dc.date.issued
2012-12
dc.identifier.citation
Forcato, Diego Oscar; Olmos Nicotra, Maria Florencia; Ortega, Nicolás Matías; Alessio, Ana Paula; Fili, Alejandro; et al.; 331 optimization of branched 25 kDa polyethylenimine for efficient gene delivery in bovine fetal fibroblasts ; Csiro Publishing; Reproduction Fertility and Development; 25; 1; 12-2012; 313
dc.identifier.issn
1031-3613
dc.identifier.uri
http://hdl.handle.net/11336/22782
dc.description.abstract
Cost-effective, highly efficient, and noncytotoxic transfection of bovine fetal fibroblasts (BFF) has proven difficult to achieve by regular chemical and physical methods. The aims of this study were to evaluate transient transfection efficiency and toxicity of commercially available branched 25 kDa polyethylenimine (25 kDa PEI, Sigma-Aldrich, St. Louis, MO, USA) and to optimize the transfection conditions leading to high percentages of PEI-transfected fibroblasts with minimum cytotoxic effects. Bovine fetal fibroblast (BFF) cells were seeded a day before transfection in 24-well plates at a density of 3 × 104 cells per well in DMEM with antibiotics and 10% SFB. When 70 to 90% confluence was reached, cells were washed with PBS and incubated in DMEM without antibiotics or SFB. For the transfection-mix preparation, increasing amounts of plasmidic DNA (pZsGreen1; 2 to 6 µg) were added to 50 µL of DMEM without antibiotics or SFB, incubated for 5 min at room temperature, and complexed with 0.5 to 4 µg of PEI (from 1 mg mL–1 solution) in 50 µL of PBS for 10 min. This transfection mix was added to the cell cultures and, 2 h later, 500 µL of DMEM with antibiotics and 10% SFB was added to each well. Detection of green fluorescent protein (GFP) expression by flow cytometry (reported as percentage of green fluorescent cells) was performed 48 h after transfection. Results were analysed by ANOVA and Tukey test and expressed as mean ± SEM (P < 0.05). We found no significant difference between the percentage of GFP-positive cells transfected with 1 or 2 µg of 25 kDa PEI at 2 µg of DNA/well (15.2 ± 1.3 v. 16.9 ± 0.9%, respectively; P > 0.05), whereas cells transfected with 1 or 2 µg of low-molecular-weight PEI (2 kDa) showed extremely low transfection efficiencies. Increasing the DNA load up to 6 µg significantly enhanced cell transfection (3.5- and 6-fold comparing 2 µg v. 4 µg and 6 µg of DNA, respectively; P < 0.05) at 1 and 2 µg of 25 kDa PEI/well. In order to evaluate the cytotoxic effect of PEI, cell viability was determined using the MTT assay in 96-well plates (cells/well), with each condition scaled down to replicate the effect of 2 kDa or 25 kDa PEI in a 24-well plate. The MTT results (expressed as % of the control) indicated that PEI became cytotoxic at concentrations equivalent to 2 and 4 µg/well (54.7 ± 3.4 and 18.5 ± 5.7, respectively), whereas 1 µg/well produced a slight detrimental effect on cell viability (90.0 ± 2.6). No evidence of cytotoxicity was observed when the BFF were incubated with 0.5 µg/well of 25 kDa PEI and 1 or 2 µg/well of 2 kDa PEI. To study if a combination of low- and high-molecular-weight PEI could improve transfection efficiency and reduce toxicity, we tested a mixture (1 : 1) of 2 kDa and 25 kDa PEI. Even though the 1 : 1 mixture was less cytotoxic, the efficiency of gene delivery was not improved. We conclude that, under our experimental conditions, the highest percentage of GFP-expressing cells with good viability was obtained when 1 µg of 25 kDa PEI was added per well. Therefore, branched 25 kDa PEI transfection represents an efficient, simple, and cost-effective alternative for gene delivery in bovine fibroblast cells in culture.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
Csiro Publishing
dc.rights
info:eu-repo/semantics/restrictedAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
Polyethylenimine
dc.subject
Fetal Fibroblast
dc.subject
Bovine
dc.subject
Gene Delivery
dc.subject.classification
Ética relacionada con Biotecnología Agrícola
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Biotecnología Agropecuaria
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CIENCIAS AGRÍCOLAS
dc.title
331 optimization of branched 25 kDa polyethylenimine for efficient gene delivery in bovine fetal fibroblasts
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2017-08-18T15:00:11Z
dc.journal.volume
25
dc.journal.number
1
dc.journal.pagination
313
dc.journal.pais
Australia
dc.description.fil
Fil: Forcato, Diego Oscar. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Olmos Nicotra, Maria Florencia. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Ortega, Nicolás Matías. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Alessio, Ana Paula. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Fili, Alejandro. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Rodriguez, Natalia Evelin. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Bosch, Pablo. Universidad Nacional de Rio Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.journal.title
Reproduction Fertility and Development
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1071/RDv25n1Ab331
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/http://www.publish.csiro.au/rd/RDv25n1Ab331


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