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Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues.

Luft C, Ketteler R - J Biomol Screen (2015)

Bottom Line: The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms.Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means.Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London, UK.

No MeSH data available.


Related in: MedlinePlus

General gene delivery mechanisms. (A) Electroporation. During electroporation, cell membranes are destabilized allowing nucleic acid entry into the cell. (B) Reagent-based techniques. The reagents used form complexes with the negatively charged nucleic acids, which are then taken up by the cell via endocytosis. Reagents include cationic lipids, cationic polymers, and calcium phosphate. Cationic lipids form liposomes, which will fuse with the cell membrane and endosomes causing the release of the nucleic acids into the cytoplasm. Cationic polymers such as polyethylenimine condense nucleic acids. They act as a proton sponge, thus buffering acidic endolysosomes and possibly causing their rupture. How calcium phosphate/DNA precipitates are taken up and released into the cytoplasm is not well understood so far. (C) Biolistic particle delivery. Nucleic acid–coated gold particles are shot at target cells. (D) Microinjection. Via an injection needle, nucleic acids can be directly delivered into the nucleus or cytoplasm. Less frequently used methods to deliver genetic material into cells like magnetofection or laserfection as well as viral transduction methods are not displayed.
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fig1-1087057115579638: General gene delivery mechanisms. (A) Electroporation. During electroporation, cell membranes are destabilized allowing nucleic acid entry into the cell. (B) Reagent-based techniques. The reagents used form complexes with the negatively charged nucleic acids, which are then taken up by the cell via endocytosis. Reagents include cationic lipids, cationic polymers, and calcium phosphate. Cationic lipids form liposomes, which will fuse with the cell membrane and endosomes causing the release of the nucleic acids into the cytoplasm. Cationic polymers such as polyethylenimine condense nucleic acids. They act as a proton sponge, thus buffering acidic endolysosomes and possibly causing their rupture. How calcium phosphate/DNA precipitates are taken up and released into the cytoplasm is not well understood so far. (C) Biolistic particle delivery. Nucleic acid–coated gold particles are shot at target cells. (D) Microinjection. Via an injection needle, nucleic acids can be directly delivered into the nucleus or cytoplasm. Less frequently used methods to deliver genetic material into cells like magnetofection or laserfection as well as viral transduction methods are not displayed.

Mentions: The cell membrane acts as a primary barrier to the entry of macromolecules into cells. From a biotechnological perspective, the uptake of large macromolecules, in particular of plasmid DNA and RNA oligonucleotides, is highly desirable for gene manipulation purposes. Accordingly, various methods have been developed that allow the introduction of exogenous material to the cell. This process is generally referred to as transfection. There are two main categories for transfection. The first is reagent-based methods including lipofection, calcium phosphate precipitation, cationic polymers, DEAE-dextran, activated dendrimers, and magnetic beads (Table 1). Reagent-based transfection methods use cellular uptake processes such as endocytosis and macropinocytosis or simply fusion of liposome particles with the plasma membrane (Fig. 1). The other method is instrument based including biolistic technologies, microinjection, laserfection/optoinjection, and electroporation (Table 1). Virus-based methods are generally termed transduction, which is highly efficient for most cell types, including stem cells.1 Transduction is mechanistically and technically very distinct from direct gene transfer by transfection as it requires the production of encapsulated DNA or RNA in virus-like particles by an intermediate step and will not be discussed here in detail. In the scope of this review, we will focus on transfection for gene manipulation and large-scale high-throughput screening (HTS) studies.


Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues.

Luft C, Ketteler R - J Biomol Screen (2015)

General gene delivery mechanisms. (A) Electroporation. During electroporation, cell membranes are destabilized allowing nucleic acid entry into the cell. (B) Reagent-based techniques. The reagents used form complexes with the negatively charged nucleic acids, which are then taken up by the cell via endocytosis. Reagents include cationic lipids, cationic polymers, and calcium phosphate. Cationic lipids form liposomes, which will fuse with the cell membrane and endosomes causing the release of the nucleic acids into the cytoplasm. Cationic polymers such as polyethylenimine condense nucleic acids. They act as a proton sponge, thus buffering acidic endolysosomes and possibly causing their rupture. How calcium phosphate/DNA precipitates are taken up and released into the cytoplasm is not well understood so far. (C) Biolistic particle delivery. Nucleic acid–coated gold particles are shot at target cells. (D) Microinjection. Via an injection needle, nucleic acids can be directly delivered into the nucleus or cytoplasm. Less frequently used methods to deliver genetic material into cells like magnetofection or laserfection as well as viral transduction methods are not displayed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4543902&req=5

fig1-1087057115579638: General gene delivery mechanisms. (A) Electroporation. During electroporation, cell membranes are destabilized allowing nucleic acid entry into the cell. (B) Reagent-based techniques. The reagents used form complexes with the negatively charged nucleic acids, which are then taken up by the cell via endocytosis. Reagents include cationic lipids, cationic polymers, and calcium phosphate. Cationic lipids form liposomes, which will fuse with the cell membrane and endosomes causing the release of the nucleic acids into the cytoplasm. Cationic polymers such as polyethylenimine condense nucleic acids. They act as a proton sponge, thus buffering acidic endolysosomes and possibly causing their rupture. How calcium phosphate/DNA precipitates are taken up and released into the cytoplasm is not well understood so far. (C) Biolistic particle delivery. Nucleic acid–coated gold particles are shot at target cells. (D) Microinjection. Via an injection needle, nucleic acids can be directly delivered into the nucleus or cytoplasm. Less frequently used methods to deliver genetic material into cells like magnetofection or laserfection as well as viral transduction methods are not displayed.
Mentions: The cell membrane acts as a primary barrier to the entry of macromolecules into cells. From a biotechnological perspective, the uptake of large macromolecules, in particular of plasmid DNA and RNA oligonucleotides, is highly desirable for gene manipulation purposes. Accordingly, various methods have been developed that allow the introduction of exogenous material to the cell. This process is generally referred to as transfection. There are two main categories for transfection. The first is reagent-based methods including lipofection, calcium phosphate precipitation, cationic polymers, DEAE-dextran, activated dendrimers, and magnetic beads (Table 1). Reagent-based transfection methods use cellular uptake processes such as endocytosis and macropinocytosis or simply fusion of liposome particles with the plasma membrane (Fig. 1). The other method is instrument based including biolistic technologies, microinjection, laserfection/optoinjection, and electroporation (Table 1). Virus-based methods are generally termed transduction, which is highly efficient for most cell types, including stem cells.1 Transduction is mechanistically and technically very distinct from direct gene transfer by transfection as it requires the production of encapsulated DNA or RNA in virus-like particles by an intermediate step and will not be discussed here in detail. In the scope of this review, we will focus on transfection for gene manipulation and large-scale high-throughput screening (HTS) studies.

Bottom Line: The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms.Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means.Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London, UK.

No MeSH data available.


Related in: MedlinePlus