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1

Influence of electroporation on chicken blastoderm cell viability in vitro

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Wawrzynska M., Bednarczyk M., Lakota P., Lubiszewska M.
Folia Biologica
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2008
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vol. 56
|
issue 3-4
197-201
EN
The aim of this study was to compare two types of devices used for blastoderm cell (BC) transfection: the Nucleofector (Amaxa, Biosystems) and the Multiporator (Eppendorf). To assess the influence of electric current on BCs, different conditions of both nucleofection and electroporation were used. Next, the viability of cells was assessed. The highest number of cells (90.8%) was viable after nucleofection in the G10 program,. After transfection in the presence of pmaxGFP, the A23 program was found to be most advantageous. The elecroporation experiment with theMultiporator (Eppendorf) showed a significant influence of osmotic pressure and voltage on BC viability. Namely, in the isoosmolar buffer BC viability was statistically higher (P#0.05) in comparison to the hypoosmolar buffer. The, viability of cells was statistically higher (P#0.05) after application of 25V as compared to 50V. The efficiency of transfection in the presence of EGFP-C1 after electroporation in 2 pulses, 25V, 500 ?s in the isoosmolar buffer was better than in the recommended conditions in the Amaxa Biosystems A23 program.
2

Transient expression assay for the optimization of direct gene transfer into cucumber meristem protoplasts by electroporation

100%
Burza W., Wochniak P., Wroblewski T., Malepszy S.
Journal of Applied Genetics
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1995
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vol. 36
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issue 1
1-10
EN
The paper obtained a new way of viable and very homogenous cucumber protoplasts.Protoplasts from cells formed in the shoot tip meristem culture were isolated from suspension.Plasmid pBI121 was introduced using imulse electric field.Effectiveness of transformation process was determinated by transient expression of ?-glucoronidase (GUS) gene, controlled by promotor 35S.The ativity of ?-glucoronidase enzyme as a product of GUS reporter gene was estimated by fluorometric method (Jefferson 1987).Parameters of electroporatian process were optimized.The transient expression of GUS gene was measured 24 h after electroporation.The highest effectiveness of transformation process was achieved using three electric impulses at the initial voltage of 250-350V at 10-sec.intervals as a results of discharging a 140 ?F capacitor and 50-70 ?g x cm/1000 plasmid DNA in the presence of 50 ?g x cm/1000 carrier DNA.The system presented is an effective method of exogenic DNA transfer, which is indicated by a high transient expression of the reporter gene.In comparison to Agrobacterium tumefaciens and A.rhizogenes, this alternative method of gene transfer can be used for obtaining transgenic cucumber plants.
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