Direct Gene Transfer Chemical Mediated

Direct Gene Transfer Chemical MediatedIntroduction: DNA is an anionic polymer, larger molecular weight, hydrophilic and sensitive to nuclease degradation in biological matrices.DNA cannot easily cross the physical barrier of membrane and enter the cells unless assistedVarious charged chemical compounds can be used to facilitate DNA transfer directly to the cell. Chemicals introduced near the vicinity of recipient cells thereby disturbing the cell membranes, widening the pore size and allowing the passage of the DNA into the cell.Ideal chemical for gene transfer?An ideal chemical used for DNA transfer should have the ability to-Protect DNA against nuclease degradation.Transport DNA to the target cells.Facilitate transport of DNA across the plasma membrane.Promote the import of DNA into the nucleus.

Commonly Used Methods Of Chemical TransfectionCalcium phosphateDEAE dextranCationic LipidOther polymers - poly-L-lysine (PLL), polyphosphoester, chitosan, dendrimers

PEG Mediated Gene Transfer

PEG Mediated Gene TransferPEG is inert & least toxic to cells/protoplastProtoplasts take ups naked DNA with PEGPEG in complex with divalent cation can disturb molecular organization of plasma membrane Ca2+ neutralizes ve charges on protoplast, PEG increases rate of endocytosis.PEG Mediated Gene TransferFactors affecting:Linearized ds-plasmid DNAMg/Ca ion conc.Disadvantage and advantages:Regeneration of fertile plants from protoplasts is a problematic for some species.The DNA used for transformation is also susceptible to degradation and rearrangement. advantages: protoplast can isolated and transformed in number of plants species.

ProtocolTo protoplast suspension add linearized plasmid DNATo this add 40% of PEG4000(w/v) dissolved in mannitol & calcium nitrate solution. pH - 8Incubation 5 minsHeat shock DNA enters the nucleus and integrates into the host genome.

Modified ProtocolAdding PEG after DNAHeat shock treatment of protoplasts at 460C followed by chilling on ice.CaCl2/PEG-stimulated DNA uptake in wheat, tobacco maize

Lipofection:Introduction of DNA in to cell via Liposome.The DNA is enclosed in a artificial lipid vesicle called liposome.Induced to fuse with protoplast using PEG.DNA + liposome enders the protoplast through endocytosisAdhesion of liposomes to protoplast surfaceFusion of liposomes at the site of adhesionRelease of plasmids inside the cell.Use of vely charged unilammellar liposome (phosphatidylserine)-DNA easily complexes with these liposome.hey may bemulti-lamellar or unilamellar vesicleswith a size range of0.1 to 10 micrometeror20-25 nanometers respectively.They can be preloaded with DNA by two common methods- membrane-membrane fusionandendocytosisthus forming DNA- liposome complex. This complexfuses with the protoplasts to release the contents into the cell. Animal cells, plant cells, bacteria, yeast protoplasts are susceptible to lipofection method.Liposomes can be classified as either cationic liposome orpH-sensitive.

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LipofectionCationic / neutral lipids from stable complex with DNA lipoplexesLiposome preparation-dispersion of phospholipids like Phosphatidyl Choline in water by sonicationCationic lipid (Dimethyl Diocta Decyl ammonium bromide, DDAB) or a synthetic cationic cholesterol derivative, along with a neutral lipid (dioleoyl-L-alpha-phosphatidyl ethanolamine, DOPE).Cationic liposome eg : Lipofectin by Gibco BRL CellFectin by Invitogen

Cationic liposomesare positively charged liposomes which associate with the negatively charged DNA molecules by electrostatic interactions forming a stable complex13Advantages:

Simplicity.Long term stability.Protection of nucleic acid from degradationApplicable to wide verity of cell types.High degree of reproducibility.Biocompatibility- reduced level of cytotoxicity

Calcium phosphate method:Method involves the formation of a fine DNA/calcium phosphate co-precipitate which first settles on the cells and then internalized by endocytosis. The precipitate must be formed freshly .The DNA escapes and reaches the nucleus and can be then expressed. This method is suitable only for actively dividing cells This method gives only 1-2% efficiency. Increasing efficiency by using Buffer and high-quality plasmid DNA + DMSO shock.

Ca/DNA coprecipitates, when formed at cell surfaces, can be taken up by cellsThis precipitate is engulfed by cells through endocytosis and the DNA gets integrated into the cell genomein plants, protoplasts are amenable to co-precipitation

Calcium phosphate co-precipitationProtocolMixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/phosphate solution (isotonic)and allowing the mixture to incubate at room temperature. This generates a precipitate (DNA-CaPO 4) that is dispersed onto the cultured protoplasts.Precipitate is taken-up by the cells via endocytosis or phagocytosis. Co-precipitation

Calcium phosphate co-precipitationAdvantagesSimple and inexpensiveApplicability to generate stably transfected cell linesHighly efficient (cell type dependent) and can be applied to a wide range of cell types.Can be used for stable or transient transfectionDisadvantagesToxic especially to primary cellsSlight change in pH, buffer salt concentration and temperature can compromise the efficacyRelatively poor transfection efficiency compared to other chemical transfection methods like lipofection.Random integration into host cell

DEAE-Dextran

Diethyl amino ethyl dextran is a soluble high molecular weight polycationic carbohydrate It associates with negatively charged nucleic acids.It promotes interactions between DNA and endocytotic machinery of the cell.

DEAE-Dextran Method:The negatively charged DNA and positively charged DEAE dextran form aggregates through electrostatic interaction and form a polyplex. A slight excess of DEAE dextran in mixture results in net positive charge in the DEAE dextran/ DNA complex formed. These complexes, when added to the cells, bind to the negatively charged plasma membrane and get internalized through endocytosis. Complexed DNA delivery with DEAE-dextran can be improved by osmotic shock using DMSO or glycerol.DEAE-Dextran AdvantagesSimple and inexpensiveMore sensitiveCan be applied to a wide range of cell typesCan be used for transient transfection.DisadvantagesToxic to cells at high concentrationsTransfection efficiency varies with cell typeCan only be used for transient transfectionbut not forstable transfectionTypically produces less than 10% delivery in primary cells.

DMSO25-30% of DMSO increases membrane permeability & enhance the uptake.Polybene + DMSO25-30% of DMSO increases membrane permeability & enhance the uptake.

Reference:Gene Cloning and DNA Analysis: an introduction by Brown TA. 2006.Biotechnology By B.D. SinghPrinciples of Gene Manipulation and Genomics by S.B. primrose and RM TwymanGene TransferTransformation: introduction of genetic materials into bacteria

Transfection: introduction of genetic materials into eukaryotic cells (e.g. fungi, plant, or animal cells)

Transduction: introduction of genetic materials using viruses

Lipofection: introduction of genetic materials using liposomes

Stable vs. Transient Gene TransferStable Gene Transfer: achieved by plasmid integration in the host genome or episomal replication of the transferred plasmid.

Transient Gene Transfer: the foreign DNA is usually not integrated into the nuclear genome and will be degraded or diluted through mitosis