Transfection method
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transfection method
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Transfection method
- 3. process of deliberately introducing nucleic acids into cells. often used for non-viral methods in eukaryotic cells. Cells that have incorporated the foreign DNA are called transfectants. Terminology Transfection: this term is reserved to describe nonviral gene delivery method in eukariotic in animal cell. Transformation: this term is reserved to describe nonviral gene delivery method in bacteria and nonanimal eukaryotic cell Transduction or infecion :is a reserved to define gene delivery method using viruses.
- 4. Type of transfection Transient transfection when DNA is not integrated into host genome but genes are expressed for a limited time( 24-96 hours(. the effect on target gene expression is temporary (24-72 hours for RNA probes, 48- 96 hours for DNA probes). used for studying: gene knockdown or silencing with inhibitory RNAs, or protein production on a small scale Stable or permanent transfection transfected genetic material is integrated into the host cell genome. used for studying: protein production on a large scale, longer-term pharmacology studies, gene therapy or research on the mechanisms of long-term genetic regulation.
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- 6. Cell type Cell health Confluency Serum time DNA quality & quantity Factors Influencing Transfection Efficiency
- 7. TRANSFECTION WORKFLOW Tissue culture count cells Resuspend cells in electroporation buffer Add nucleic acid Transfect cells Plate cells Analysis Microscopy Gene ExpressionProtein Expression Western blot analysis Reporter gene activity Flow cytometry Real time PCR
- 8. Common transfection methods • Electroporation • Microinjection • Biolistic Particle Delivery • Laserfection/optoinjection Instrument (physical) based method • Cationic Lipids : Liposome/Lipoplexes • Calcium phosphate • Cationic polymer • DEAE-Dextran • Magnet-mediated transfection • Activated dendrimers Reagent (Chemical)- based method • Viral method Biological based method
- 9. a complex with overall positive charge, allowing it to interact with negatively charge cell membrane and promote uptake by endocytosis as lipophilic complex that fuses with the cell membrane and deposits the transgene directly into the cytoplasm
- 10. Electrostatic interactions between the positive charges of cationic lipid head groups and the negatively charged phosphates of the DNA backbone are the main forces that allow DNA to spontaneously associate with cationic lipids. the cationic lipid is mixed with a neutral lipid such as L-dioleoylphosphatidylethanolamine(DOPE)which can enhance the gene transfer ability of certain synthetic cationic lipids Cationic Lipids
- 11. The successful use of liposome compounds is dependent upon several factors including lipid formulation, particle size and the method of liposome preparation. An overall net positive charge of the complex allows closer association of the complex with the negatively charged cell membrane. Entry of the liposome complex into the cell: endocytosis or fusion with the plasma membrane via the lipid moieties of the liposome When liposomes encounter nucleic acids they re-form into nucleic acid lipid complexes called lipoplexes which can be actively taken up by eukaryotic cells by means of endocytosis
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- 13. Advantages of lipids: Deliver nucleic acids with high efficiency Easy to use, minimal steps required. Works in a wide variety of eukaryotic cells can be used for in vivo transfer of DNA and RNA to animals and humans successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes Disadvantages of lipids: Not applicable to all cell types. low efficiencies in most primary cells Pros and cons cationic lipid
- 14. Principle DNA is mixed with calcium chloride Addition to buffered saline/phosphate solution and incubating at room temperature Formation of DNA-calcium phosphate coprecipitates which adhere to surface of cells Uptake presumably by endocytosis or phagocytosis Calcium phosphate
- 15. Advantages: easily available Inexpensive Can be applied to wide range of cell types Can be used for transient and stable transfection Calcium phosphate appears to provide protection against intracellular and serum nucleases. Disadvantages: low efficiency is not suited for in vivo gene transfer to whole animals Size and quality of the precipitate are crucial to the success of transfection toxicity, especially to primary cells its sensitivity to slight changes in pH, temperature and buffer salt concentrations. Pros and Cons Calcium phosphate
- 16. Cationic polymer differ from cationic lipid in that they do not contain a hydrophobic moiety and are completely soluble in water. Cationic polymer cationic polymer include: polybrene, polyethyleneimine(PEI) and dendrimers. ability of cationic polymer more efficiently condense DNA. cationic polymer cannot release their DNA load into the cytoplasm Cationic polymer synthesized (lengths , geomrtry)
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- 19. Dendrimers are highly branched, globular macromolecules that are capable of interacting with DNA to form small complexes. Positively charged amino groups (termini) on the surface of the dendrimer molecule interact with the negatively charged phosphate groups of the DNA molecule to form a DNA- dendrimer complex. Complexes bound to the cell surface are taken into the cell by nonspecific endocytosis. Dendrimers
- 20. Amino groups on the dendrimers that are unprotonated at neutral pH can become protonated in the acidic environment of the endosome. This leads to buffering of the endosome, which inhibits pH- dependent endosomal nucleases. Dendrimers are stable in biological liquids and are not sensitive to temperature. highly efficient tools for tissue culture transfections. However, dendrimers are also The downside of dendrimers non- biodegradable and may cause significant cytotoxicity.
- 21. DEAE-dextran was the first non-viral transfection method verified by Vaheri and Pagano in 1965. DEAE-Dextran is a cationic polymer that tightly associates with the negatively charges nucleic acids. principle DNA is mixed with DEAE-dextran DNA/polymer complex comes into contact with negatively charged membrane due to excess of positive charge contributed by polymer entry in to the cytoplasm via endocytosis or osmotic shock induced by DMSO or glycerol. Uptake presumably by endocytosis DEAE-Dextran chemical method
- 22. Advantages: Inexpensive Easy to perform and quick Can be applied to a wide range of cell types. Low cost Disadvantages: High concentration of DEAE-Dextran can be toxic to the cell Transfection efficiency will vary with cell type Can be used only for transient transfection Typically produces less than 10% delivery in primery cells Pros and Cons DEAE-Dextran
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- 24. Magnet-mediated transfection (Magnetofection) Magnet-mediated method uses magnetic force to deliver DNA into the target cells. Nucleic acids are first associated with magnetic nanoparticles. DNA are bound to MNPs, which then move from the media to the cell surface by applying a magnetic force.
- 25. Plasmid DNA or siRNA is attached to magnet nanoparticles and incubated with cells in culture. (i) An oscillating magnet array below the surface of the cell culture plate pulls the particle into contact with the cell membrane (ii) and drags the particles from side-to-side across the cells (iii)mechanically stimulating endocytosis (iv) Once the particle/DNA complex is endocytosed, proton sponge effects rupture the endosome (v) releasing the DNA (vi) which then transcribes the target protein.
- 26. Advantages Rapid Increased transfection efficiency by direct transport, especially for low amount of nucleic acid High transfection rates for adherent mammalian cell lines and primary cell cultures Can also be performed in the presence of serum Disadvantages Require adherent cells; suspension cells need to be immobilized or centrifuged. Pros and cons Magnet-mediated transfection
- 27. in this method, naked DNA is deposited directly into the cell by exploiting a physical force Instrument(Physical )-based method
- 28. Electroporation was first reported for gene transfer studies in mouse cells . This technique is often used for cell types such as plant protoplasts. Electroporation uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells. More modern instrumentation successful transfer of DNA and RNA to primary and stem cells. Electroporation
- 29. ectroporation 1)Electroporation exposes a cell to a high-intensity electric field that temporarily destabilizes the membrane 2)During this time the membrane is highly permeable to exogenous molecules present in the surrounding media 3) DNA then moves into the cell through these holes 4)When the field is turned off, the pores in the membrane reseal, enclosing the DNA inside
- 30. Advantages fast doesn't seem to alter the biological structure or function of the target cells Easy to perform High efficiency Can be applied to transient and stable transfection a wide range of cell types Disadvantages Cell mortality low efficiency in primary cells and high mortality rates, caused by the high voltage pulses or only partially successful membrane repair. Pros and cons electroporation
- 31. Cell squeezing is a method invented in 2012 by Armon Sharei, Robert Langer and Klavs Jensen. Cell squeezing
- 32. It eliminates the possibility of toxicity or off-target effects as it does not rely on exogenous materials or electrical fields. describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells.
- 34. involves projecting microscopic heavy-metal particles (often gold or tungsten) coated with nucleic acids into recipient cells at high velocity using a biolistic device (i.e., “gene gun”). Biolistic particle delivery can be used to transiently transfect dividing and non-dividing cells in culture as well as cells in vivo, and it is often used for genetic vaccination and agriculture applications. particle bombardment))Biolistic Particle Delivery Delivery of nucleic acids into cells via high-velocity nucleic acid- coated microparticles
- 36. PDS-1000/He Biolistic Particle Delivery System
- 37. Pros and cons Biolistic particle delivery Advantages Simple, rapid technique Cell type independent Uses small amounts of DNA Can deliver large DNA fragments Requires little manipulation of cells High reproducibility Dis advantages Generally lower efficiency compared to electroporation or viral or lipid mediated transfection Requires the preparation of microparticles Instrument cost causes physical damage to the samples, and necessitates high cell numbers due to high mortality.
- 38. delivers nucleic acids into the cytoplasm or the nucleus one cell at a time by means of a fine needle; therefore, this method is limited to ex vivo applications such as the transfer of genes into oocytes to engineer transgenic animals or the delivery of artificial chromosomes. Microinjection
- 39. Advantage: Frequency of stable integration of DNA is far better as compare to other methods. Method is effective in transforming primary cells The DNA injected in this process is subjected to less extensive modifications. used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms Disadvantage: Costly. Skilled personal required. Method is useful for protoplasts and not for the walled cells. Pros and cons
- 40. This procedure uses laser light to transiently permeabilize a large number of cells in a very short time. Various substances, including ions, small molecules, (siRNAs), plasmids, proteins, can be efficiently optoinjected into numerous cell types. When the laser induces a pore in the membrane, the osmotic difference between the medium and the cytosol facilitates the entry of nucleic acids or other desired substances in the medium into the cell. Laserfection/Optoinjection
- 41. Pros and cons Advantage high transfection efficiency and the ability to make pores at any location on the cell. Work with many cell type Fewer cell maniupullation needed Disadvantage requires an expensive laser-microscope system and the cells to be attached to a substrate.
- 42. Comparison of Different Gene Transfer Methods
- 43. Comparison of Different Gene Transfer Methods
- 44. A Comparison of Different Viral Systems
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Notas do Editor
- i.e., introduction of DNA (or RNA) from a prokaryote-infecting virus or bacteriophage into cells, resulting in an infection Transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane
- Thus. Transient transfection with mRNA can deliver even more rapid results than with conventional plasmid DNA, because mRNA can be expressed outside of the nucleus; in some systems, it is possible for transfected mRNA to be expressed only minutes after transfection. Although the production of large amounts of recombinant proteins with proper folding and post-translational modifications (which is not available when expressing recombinant proteins in bacterial cells) traditionally has been limited to stably transfected cells, recent advances in transient transfection and cell culture methods are changing this. Ubiquitous cell culture lines such as HEK293 and CHO cells, modified to grow in suspension rather than in plated culture conditions, can now be transiently transfected to produce large amounts of recombinant proteins, as well.
- Frequently used selectable markers are the genes encoding aminoglycoside phosphotransferase (APH; neoR gene) or hygromycin B phosphotransferase (HPH). Other selectable markers are the genes encoding adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hymidine kinase (TK), or xanthine-guanine phosphoribosyl transferase (XGPRT; gpt gene).
- Every cell line has optimal culture conditions. Refer to the American Type Culture Collection [ATCC] web site. For example, one reagent may work well with HEK-293 cells, but a second reagent is a better choice when using HepG2 cells. Promega offers several online resources to help identify a transfection reagent and protocol for your cell line, including the Transfection Assistant (www.promega.com/transfectionasst/) and FuGENE ® HD Protocol Database (www.promega.com /techserv/tools/FugeneHD/). A drop-down menu allows you to search the databases by cell line, transfection type and transfection reagent.
- TISSUE CULTURE Most mammalian cells are adherent Cultured in plates or flasks Grow in monolayer on specially treated surfaces Medium supplemented with 5-10% Fetal Calf Serum Laminar flow cabinet CO2 incubator
- ; DNA will be taken from the surroundings when the DNA is presented as a synthetic complex either as;
- Such lipids form liposomes that associate with DNA and merge with the cell membrane to enable the intracellular transfer of DNA3. Cationic transfection lipids consist of a positively charged head group, such as an amine, a flexible linker group such as an ester or ether, and two or more hydrophobic tail groups. The ability of a lipid to destroy endosomes is one of the main characteristics of a transfection reagent. . For cultured cells, an overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efficiencies, presumably because this allows closer association of the complex with the negatively charged cell membrane. Entry of the liposome complex into the cell may occur by endocytosis or fusion with the plasma membrane via the lipid moieties of the liposome Following cellular internalization, the complexes appear in the endosomes and later in the nucleus. It is unclear how the nucleic acids are released from the endosomes and lysosomes and traverse the nuclear membrane \ DOPE is considered a “fusogenic” lipid (Farhood et al. 1995), and its role may be to release these complexes from endosomes as well as to facilitate fusion of the outer cell membrane with liposome/nucleic acid complexes. While DNA will need to enter the nucleus, the cytoplasm is the site of action for RNA, protein or antisense oligonucleotides delivered via liposomes. The endosomal structure is destroyed by increasing the osmotic pressure created by the lipids' buffering action within the endosomes and by the fusion of the lipid with the endosomal membrane.
- The successful use of liposome compounds to deliver exogenous nucleic acids to specific biological system is dependent upon several factors including lipid formulation, charge ratio, particle size and the method of liposome preparation. Neutral “helper” lipids, such as DOPE, allow entrapped DNA to escape the endosomes by fusion of the liposome with the membrane.
- The cells will be treated with lysozyme to remove cell wall, resulting in protoplasts that will be centrifuged gently onto a monolayer of mammalian cells to promote fusion among them using polyethylene glycol. Using lyposomes is more commonly used for this type of transfection. This method is commonly known as lipofection. This method is far more efficient than chemical transfection method. With this method up to 90% of cells in culture dish can be transected.
- The process of transfection involves the admixture of isolated DNA (10-100ug) with solution of calcium chloride and potassium phosphate under condition which allow the precipitate of CaPO4 to be formed. In general, the following two protocols are used by the majority as a starting point. The first one relies on the calcium phosphate–DNA complex formed before the addition to the cells, and the second one relies on the slow formation of calcium phosphate–DNA complex while present in the cell culture. 4Additional modifications to the calcium phosphate method that can achieve higher efficiencies include glycerol shock and/or chloroquine treatmen
- this method gives only 1-2% transfection efficiency
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- When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications
- واحد فشار هوا بوده و مخفف پوند بر اینچ مربع میباشد و هر 14.5psi آن 1 بار میباشد . (برای تبدیل کلیه واحدهای فشار میتوانید از جدول تبدیل فشارها در بخش مقالات استفاده کنید)
- For in vitro, ex vivo (and in vivo for some plants and microbes) Applications for animal cell and organ culture, plant cell culture and explants, pollen, insects, algae, fungi and bacteria Pressure range 450–2200 psi gives flexibility and penetration — ideal for plant applications Large target area — more cells can be transformed
- First - you need the gene of interest in the proper form. A linear transgene construct is made, which contains: the structural gene of interest, with introns a strong mouse gene promoter and enhancer to allow the gene to be expressed