Springer protocols feed by Genetics/Genomics 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.A Sensible Technique to Detect Mollicutes Impurities in Human Cells Cultured in GMP Condition
In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news
Electroporation-Mediated siRNA Delivery into Tumors
Electroporation-mediated gene transfer (electro-transfection) is a powerful tool to introduce nucleic acid compounds such as plasmid DNAs, antisense oligonucleotides, and short interfering RNAs (siRNAs) into the cells. Electro-transfection is a physical gene transfer method that utilizes an electrostatic field generated with an electroporator apparatus. Here, we demonstrate a practical protocol for electro-transfection (electro-delivery) of siRNA into cells in vivo and further demonstrate the application of the method to cancer therapy. We successfully developed an original electrode (the plate and fork-type electrode) and...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Direct Imaging of siRNA Electrotransfer at the Single-Cell Level
In this study, we investigated, by direct visualization at the single-cell level, the electro-delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time-lapse fluorescence microscopy and was correlated with the silencing of EGFP expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. The oligonucleotide then freely diffused across the cytosol. Therefore, we show that th...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Gold Nanoparticle-Enhanced Electroporation for Leukemia Cell Transfection
Electroporation serves as an attractive nonviral gene delivery approach for its effectiveness, operational simplicity, and no restrictions of probe or cell type. The commercial electroporation systems have been widely adopted in research and clinics with protocols usually compromising appropriate transfection efficiency and cell viability. By introducing gold nanoparticles (AuNPs), we demonstrated greatly enhanced performance of electroporation from two aspects: the highly conductive, naked AuNPs help reduce the potential drop consumed by the electroporation solution so that the majority of the applied voltage of an electr...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Short-Fragment DNA-Mediated In Vivo DNA Electroporation Delivery
Electroporation is an effective physical delivery method. A variety of factors have been shown to affect the electroporation-mediated gene delivery efficiency. Here we report the usefulness of noncoding short-fragment DNA (sf-DNA) for facilitating electroporation-mediated gene transfer. The plasmid pGL3-control encoding firefly luciferase was injected into tissue together with or without sf-DNA in different length or dose. Immediately after injection, the tissues were electroporated and the level of luciferase activity was assessed 24 h later. The results showed that plasmid DNA formulated with sf-DNA resulted in significa...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Electroporation Formulation for Cell Therapy
Cell transfection efficiency often determines the success of cell-based gene therapy. Cell transfection via Nucleofector technology yields high transfection efficiency and low cytotoxicity. However, owing to trade secrecy, the components in each buffer are unknown, which not only increases the cost of electroporation studies but also limits the application of Nucleofector in clinical cell-based gene therapies. Thus, we developed a three-step method to determine the optimal conditions, including buffer, program, and additional polymer, in electroporation for multiple cancers and stem cell lines. This method could reduce the...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
The Impact of Non-electrical Factors on Electrical Gene Transfer
Electrical pulses directly and effectively boost both in vitro and in vivo gene transfer, but this process is greatly affected by non-electrical factors that exist during electroporation. These factors include, but are not limited to, the types of cells or tissues used, property of DNA, DNA formulation, and expressed protein. In this mini-review, we only describe and discuss a summary of DNA properties and selected DNA formulations on gene transfer via electroporation. The properties of DNA were selected for review because a substantial amount of remarkable work has been performed during the past few years but has received...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Electropermeabilization of the Cell Membrane
Membrane electropermeabilization is the observation that the permeability of a cell membrane can be transiently increased when a micro-millisecond external electric field pulse is applied on a cell suspension or on a tissue. Applicative aspects for the transfer of foreign molecules (macromolecules) into the cytoplasm are routinely used. But only a limited knowledge about what is really occurring in the cell and its membranes at the molecular levels is available. This chapter is a critical attempt to report the present state of the art and to point out some of the still open problems. The experimental facts associated to me...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Electroporation-Based Gene Therapy: Recent Evolution in the Mechanism Description and Technology Developments
Thirty years after the publication of the first report on gene electrotransfer in cultured cells by the delivery of delivering electric pulses, this technology is starting to be applied to humans. In 2008, at the time of the publication of the first edition of this book, reversible cell electroporation for gene transfer and gene therapy (nucleic acids electrotransfer) was at a cross roads in its development. In 5 years, basic and applied developments have brought gene electrotransfer into a new status. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the c...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
A Yeast-Based Recombination Assay for Homing Endonuclease Activity
Homing endonucleases (HEs) are natural enzymes that cleave long DNA target with a high specificity and trigger homologous recombination at the exact site of the break. Such mechanisms can thus be used for all the applications covered today by the generic name of “genome engineering”: targeted sequence insertion, removal, or editing. However, before being able to address those applications, the engineering of HEs must be mastered so that any potential target would be efficiently and specifically recognized and cleaved. Working on the I-CreI model, we have developed a very powerful platform to generate HEs with n...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Rapid Screening of Endonuclease Target Site Preference Using a Modified Bacterial Two-Plasmid Selection
Homing endonucleases and other site-specific endonucleases have potential applications in genome editing, yet efficient targeting requires a thorough understanding of DNA-sequence specificity. Here, we describe a modified two-plasmid genetic selection in Escherichia coli that allows rapid profiling of nucleotide substitutions within a target site of given endonucleases. The selection utilizes a toxic plasmid (pTox) that encodes a DNA gyrase toxin in addition to the endonuclease target site. Cleavage of the toxic plasmid by an endonuclease expressed from a second plasmid (pEndo) facilitates growth under selective conditions...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
A Two-Plasmid Bacterial Selection System for Characterization and Engineering of Homing Endonucleases
Homing endonucleases recognize long DNA sequences and generate site-specific DNA double-stranded breaks. They can serve as a powerful genomic modification tool in various industrial and biomedical applications. Here, we describe a two-plasmid bacterial selection system for characterization and engineering of homing endonucleases. This selection system couples the DNA cleavage activity of a homing endonuclease with the survival of host cells. Therefore, it can be used for assaying in vivo activity of homing endonucleases. Moreover, due to its high sensitivity, it can be applied for directed evolution of homing endonucleases...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
PCR Analysis of Chloroplast Double-Strand Break (DSB) Repair Products Induced by I-CreII in Chlamydomonas and Arabidopsis
Homing endonuclease I-CreII has been used to study the consequences and repair of a double-strand break (DSB) in the chloroplast genome of Chlamydomonas and Arabidopsis. Since I-CreII is from a mobile psbA intron of Chlamydomonas, it cleaves the psbA gene of an intronless-psbA strain of Chlamydomonas. And it cleaves specifically in the psbA gene of Arabidopsis, which is naturally intronless. We have shown further that most of the repair products of an I-CreII-induced break in chloroplast DNA can be defined by PCR analysis with total nucleic acids and the appropriate primers. Here, we provide protocols for small-scale prepa...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Mapping Free-Standing Homing Endonuclease Promoters Using 5′RLM-RACE
5′RLM-RACE is a PCR-based technique used to map the 5′ termini of transcripts in both eukaryotic and prokaryotic organisms. Free-standing homing endonuclease promoters often lack recognizable promoters making predicting the transcriptional start site challenging. Furthermore, homing endonucleases are often expressed at very low levels making transcript mapping a challenge. Here, I present a 5′RLM-RACE protocol with special considerations for the expected abundance of homing endonucleases and for their potential to be subjected to RNA processing events. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein
Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
