ibidi Application Guide Transfection The Principle of Transfection . 2 Transfection Methods . 3 Chemical Transfection . 3 Lipofection . 3 Membrane Fusion . 4 Membrane Fusion vs . Lipofection . 4 Calcium Phosphate . 5 Cationic Polymers . 5 Viral Transduction . 6 Adenoviral Transduction . 6 Lentiviral Transduction . 7 Method Comparison . 7 Selected Publications Physical Transfection . 8 F. Martin-Sanchez et al. Inflammasome-dependent IL-1[beta] release depends upon membrane permeabilisation. Cell Death Microinjection . 8 Differ, 2016, 10.1038/cdd.2015.176 Electroporation . 8 M. Moch et al. Effects of Plectin Depletion on Keratin Network Dynamics and Organization. PloS one, 2016, 10.1371/journal. Experimental Example . 9 pone.0149106 G. Burgstaller, S. Vierkotten, M. Lindner, M. Königshoff and O. Eickelberg. Multidimensional immunolabeling and 4D time- lapse imaging of vital ex vivo lung tissue. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2015, 10.1152/ajplung.00061.2015 A. Csiszar, N. Hersch, S. Dieluweit, R. Biehl, R. Merkel and B. Hoffmann. Novel fusogenic liposomes for fluorescent cell labeling and membrane modification. Bioconjugate chemistry, 2010, 10.1021/bc900470y C. Kleusch, N. Hersch, B. Hoffmann, R. Merkel and A. Csiszár. Fluorescent lipids: Functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization. Molecules, 2012, 10.3390/molecules17011055 .com The Principle of Transfection siRNA Fuse-It-siRNA mRNA Virus Particle Fuse-It-mRNA Adenoviral Transduction CAR Membrane Fusion Endosome ds-siRNA ssRNA RELEASE OF RNA Protein PROTEIN REVERSE Protein EXPRESSION ss-siRNA TRANSCRIPTION DEGRADATION TRANSLATION RELEASE dsDNA OF DNA TRANSLATION mRNA Lentiviral RISC mRNA Transduction Virus Particle Protein STABLE TRANSCRIPTION INTEGRATION TRANSLATION mRNA LYSOSOMAL DEGRADATION Microinjection Lipofection RELEASE OF DNA Plasmid Plasmid DNA DNA Lysosome Transfection – Reagent – + Endosome – + + Plasmid DNA Electroporation Plasmid DNA Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Calcium Ca2+ Phospate Ca2+ Ca2+ + + Plasmid + + DNA Calcium + + + Phospate + + + Cationic Cationic + Polymers Polymers Transfection is defined as the process of inserting nucleic acids (e g. ,. plasmid DNA, cDNA, mRNA, miRNA, siRNA) into the cytoplasm of eukaryotic cells . In addition, proteins and nanoparticles such as beads or dyes can be transfected . In the field of cell biology, transfection is an important and widely used tool for analyzing the function of various genes . This method can be used for gene silencing via RNAi, gene editing via CRISPR/Cas9, as well as overexpression studies by cellular integration of plasmid DNA, mRNA, or proteins . Many researchers conduct transfection experiments on a daily basis, which requires a concise understanding of the biological background, precise and detailed planning, and a robust transfection protocol . Kim T.K., Eberwine J.H. Mammalian cell transfection: The present and the future. Anal Bioanal Chem, 2010, 10.1007/ s00216-010-3821-6 2 Transfection Methods Naturally, cells usually do not take up foreign nucleic acids without external stimulation . Thus, an efficient and biocompatible transfection method is needed, which has to be accurately adapted to the cell type and the material to be transferred . Many transfection methods have been developed, which can be basically subdivided into three different groups: chemical transfection, physical transfection, and viral transduction . In this Application Guide, you can find a brief description of the most commonly applied methods, summarizing the characteristics, advantages, and pitfalls of each approach . Chemical Transfection Viral Transduction Physical Transfection • Lipofection • Adenoviral Transduction • Microinjection • Membrane Fusion • Lentiviral Transduction • Electroporation • Calcium Phosphate • Cationic Polymers Chemical Transfection Lipofection Protein Applications: TRANSCRIPTION TRANSLATION mRNA Lipofection is commonly used to transfer nucleic LYSOSOMAL acids such as RNA or DNA into eukaryotic cells . After DEGRADATION Lipofection RELEASE protocol optimization, this method is easy to apply OF DNA and yields highly reproducible results of transient Plasmid DNA Lysosome and stable transfections . However, lipofection Transfection efficiency strongly depends on the cell type and has Reagent to be tested and optimized in advance . Especially Endosome for primary and non-dividing cells, the viability after the transfection process might be decreased due to the high cellular sensitivity . In contrast to membrane fusion, lipofection is based on endosomal molecule uptake, which might delay the time of analysis . ibidi Solutions: Principle: The ibidi µ-Slides, µ-Dishes, and µ-Plates are ideally suited for lipofection using various Usually, a mixture of neutral and cationic liposomes cell numbers and volumes . Volume calculations forms complexes with the nucleotides of interest . for frequently used sizes are available in the These complexes pass the cell membrane and instructions of each Torpedo reagent . then release the nucleotides into the cytoplasm via endocytosis . Then, the nucleotides either successfully The pCMV/pCAG-LifeAct plasmid can be used escape the endosome or undergo lysosomal for non-toxic F-actin visualization in living cells . degradation, of which the latter might lead to reduced transfection efficiency . After endosomal escape, the nucleotide is expressed in the target cells . Felgner P.L., et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. 1987, Proc Natl Acad Sci U S A, 10.1073/pnas.84.21.7413 3 Membrane Fusion Fusogenic Applications: Liposome Membrane fusion is a novel and highly superior transfection method to incorporate various molecules and particles into mammalian cells . Not only cells that undergo cell division, but also primary and non-dividing Liposomal Carrier cells can be transfected by using this innovative method . It allows for highly efficient transfer of mRNA, siRNA, proteins, beads, dyes, and other molecules . Since the reagents are non-toxic to sensitive and FUSION difficult-to-transfect cells, even primary neurons, keratinocytes, and stem cells retain high viability after the transfection . Additionally, the very short incubation times minimize cell stress and provide an almost unaffected cell behavior after the molecule transfer . Principle: IMMEDIATE RELEASE INTO THE CYTOPLASM Liposomal carriers, which consist of neutral and cationic lipids, are used to transfer the molecule of interest via membrane fusion into the cell . The molecules are first incorporated into the liposomal carriers, which upon contact, instantly fuse with the cell membrane . The included molecules are then directly released into the ibidi Solutions: cytoplasm without processes such as endocytosis and lysosomal degradation . This results in fusion The ibidi µ-Slides, µ-Dishes, and µ-Plates are efficiencies up to 80–100% within only seconds to a ideally suited for membrane fusion using various few minutes, depending on the cell type . cell numbers and volumes . Volume calculations for frequently used sizes are available in the Csiszár, A., et al. Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification. Bioconjug Chem, instructions of each Fuse-It reagent . 2010, 10.1021/bc900470y Membrane Fusion vs . Lipofection Membrane Fusion Is FusogenicFusogenic EndocytoticEndocytotic Superior to Lipofection LiposomeLiposome LiposomeLiposome In contrast to lipofection, membrane fusion does not LiposomalLiposomal LipoplexLipoplex involve endocytosis . Instead, CarrierCarrier the fusogenic liposomal carrier MembraneMembrane Fusion Fusion LipofectionLipofection simply fuses with the cell FUSIONFUSION membrane, then releases the included molecule of interest ENDOCYTOSISENDOCYTOSIS directly into the cytoplasm . This results in immediate IMMEDIATEIMMEDIATE RELEASE RELEASE and efficient transfer without INTOINTO THE THE CYTOPLASM CYTOPLASM lysosomal degradation . EndosomeEndosome ENDOSOMALENDOSOMAL RELEASERELEASE LysosomeLysosome LYSOSOMALLYSOSOMAL DEGRADATIONDEGRADATION 4 Calcium Phosphate Applications: Protein TRANSCRIPTION The calcium phosphate transfection is an inexpensive TRANSLATION mRNA and simple method for transient or stable nucleotide LYSOSOMAL DEGRADATION transfer into most cell lines .The transfection efficiency, RELEASE however, strongly depends on the cell constitution, Calcium OF DNA Phospate the pH, and the quality and the amount of the used Lysosome nucleotides . Therefore, the optimal experimental Plasmid conditions have to be tediously established in ad- DNA Ca2+ Ca2+ Ca2+ Ca2+ vance . Further, the calcium phosphate transfection Ca2+ Ca2+ Endosome Calcium Ca2+ is toxic and therefore not suitable for most sensitive Phospate Ca2+ primary cell lines . Ca2+ Principle: A mixture of the nucleotides, calcium, and phosphate buffer forms a precipitate that is taken up by the cells ibidi Solutions: via endocytosis . Then, the nucleotides either escape the endosome or undergo lysosomal degradation, of The ibidi µ-Slides, µ-Dishes, and µ-Plates are which the latter might lead to reduced transfection ideally suited for calcium phosphate transfection efficiency . Successfully escaped nucleotides can with various cell numbers . then be expressed in the target cells . Cationic Polymers Applications: Protein TRANSCRIPTION Using cationic polymers, nucleotides can be tran- TRANSLATION mRNA siently transfected into eukaryotic cells in an LYSOSOMAL inexpensive