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Ef?cient delivery of siRNA into mouse preimplantation embryos by electroporation

Bohao Chang,Hui Peng,Jinhai Tian,Jianmin Su,Hengde Zhang,Xueyao Bai,Yong Zhang,

生物工程学报 , 2012,
Abstract: We developed a detailed electroporation method to deliver efficiently siRNA into mouse preimplantation embryos. By introducing Cy3 labeled negative control small interfering RNA (siRNA) into mouse preimplantation embryos, we optimized conditions for the electroporation, including the voltage, pulse duration, pulse number, electroporation buffer and an important step to weaken the zona pellucida. Embryonic survival rate, transfection rate and blastocyst development rate were evaluated under the converted fluorescence microscope, by embryos counting and statistical analysis. The best transfection was achieved in opti-MEM under the conditions of 30 V, 1 ms, 3 pulses, and the duration of digestion in tyrode's solution was 10 s. In conclusion, the proposed electroporation approach here is a simple and efficient tool to deliver siRNA for RNA interference (RNAi) into mouse preimplantation embryos.
Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts  [cached]
Amir Amiri Yekta,Azam Dalman,Mohammad Hossein Sanati,Nayeralsadat Fatemi
Cell Journal , 2013,
Abstract: Objective: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.Materials and Methods: In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 μg DNA per pulse in the first experiment and increments of 10 μg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR)-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.Results: The results showed no significant difference among three first concentrations except for group 1 (10 μg/pulse) and group 3 (30 μg/pulse), but there was a significant difference between these groups and the fourth group (p<0.05), as this group (40 μg/pulse) statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH) results indicated the transgene was integrated in a single position in PCR positive cells.Conclusion: Increasing amount of using the vector 40μg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency.
Optimal Electroporation Condition for Small Interfering RNA Transfection into MDA-MB-468 Cell Line
Rita Arabsolghar,Mozhgan Rasti
Iranian Journal of Medical Sciences , 2012,
Abstract: Background: Electroporation is a valuable tool for small interfering RNA (siRNA) delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA. Methods: To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection. Results: The best condition for electroporation of this cell line was 220 volt and 975 μF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively. Conclusion: Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability.
Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal  [PDF]
Torsten Steinbrunn, Manik Chatterjee, Ralf C. Bargou, Thorsten Stühmer
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0097443
Abstract: Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in “hard-to-transfect” MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs.
Electroporation by nucleofector is the best nonviral transfection technique in human endothelial and smooth muscle cells  [cached]
Iversen Nina,Birkenes Baard,Torsdalen Kari,Djurovic Srdjan
Genetic Vaccines and Therapy , 2005, DOI: 10.1186/1479-0556-3-2
Abstract: Background The aim of this study was to determine the optimal non-viral transfection method for use in human smooth muscle cells (SMC) and endothelial cells (EC). Methods Coronary Artery (CoA) and Aortic (Ao) SMC and EC were transfected with a reporter plasmid, encoding chloramphenicol acetyltransferase type 1 (CAT), with seven different transfection reagents, two electroporation methods and a photochemical internalization (PCI) method. CAT determination provided information regarding transfection efficiency and total protein measurement was used to reflect the toxicity of each method. Results Electroporation via the nucleofector machine was the most effective method tested. It exhibited a 10 to 20 fold (for SMC and EC, respectively) increase in transfection efficiency in comparison to the lipofection method combined with acceptable toxicity. FuGene 6 and Lipofectamine PLUS were the preferred transfection reagents tested and resulted in 2 to 60 fold higher transfection efficiency in comparison to the PCI which was the least effective method. Conclusion This study indicates that electroporation via the nucleofector machine is the preferred non-viral method for in vitro transfection of both human aortic and coronary artery SMC and EC. It may be very useful in gene expression studies in the field of vascular biology. Through improved gene transfer, non-viral transfer techniques may also play an increasingly important role in delivering genes to SMC and EC in relevant disease states.
Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues  [cached]
Jean-Luc Thomas
Journal of Insect Science , 2003,
Abstract: There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos,and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473-479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B.mori embryos. By injecting the DNA solution into the hemocoel of late embryos that were finishing organogenesis, we expressed marker genes in numerous tissues following electroporation. With some adaptation of the method this was also achieved for early embryos lacking a hemocoel. Some larval tissues were also transfected. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is a reliable technique for transfecting B.mori tissues.
Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine  [PDF]
Jennifer A. Fortune,Tatiana I. Novobrantseva,Alexander M. Klibanov
Journal of Drug Delivery , 2011, DOI: 10.1155/2011/204058
Abstract: We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp.) while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.
Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution
Maja Cemazar, Ian Wilson, Gabi U Dachs, Gillian M Tozer, Gregor Sersa
BMC Cancer , 2004, DOI: 10.1186/1471-2407-4-81
Abstract: Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP) and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared.Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses.Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous distribution of gene expression in the syngeneic P22 rat tumor model.Despite some promising early results, gene therapy does not, as yet, live up to expectation [1]. The main stumbling block remains gene delivery, and all advances in the control of gene expression and selection of therapeutic genes are hampered by inefficient gene transfection. Hence the development of a safe and effective method of gene delivery in vivo is of utmost importance if gene therapy is to move from the experimental to the clinical stage.Electroporation is currently receiving much attention as a way to increase drug and DNA delivery [2-5]. Electroporation has long been used as an effective in vitro gene delivery system in both prokaryotes and eukaryotic cells. Electroporation is a physical means of importing small molecules and macromolecules into cells via increased cell membrane permeability. Electr
Comparison of the absolute and relative efficiencies of electroporation-based transfection protocols for Plasmodium falciparum
Sandra Hasenkamp, Karen T Russell, Paul Horrocks
Malaria Journal , 2012, DOI: 10.1186/1475-2875-11-210
Abstract: A time-course of luciferase reporter expression is used to provide an objective quantitative analysis of the absolute efficiency of three electroporation techniques; direct electroporation of ring stage infected erythrocytes, preloading of erythrocytes and a novel “double-tap” protocol that combines both approaches.Preloading of erythrocytes shows a mean efficiency of 9.59x10-6, some 5–180 fold more efficient than matched experiments utilizing the “double-tap” and direct electroporation of ring stage infected erythrocytes alone, respectively.Evidence presented here provides the first quantitative assessment of both the absolute and relative efficiencies of a key molecular tool used to study the biology and pathogenesis of this important human pathogen.
mRNA Transfection of Mouse and Human Neural Stem Cell Cultures  [PDF]
Samuel McLenachan, Dan Zhang, Ana Belén Alvarez Palomo, Michael J. Edel, Fred K. Chen
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083596
Abstract: The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.
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