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Transformation, Transduction and Transfection –Gene transfer methods
#1
This is post no. 1 under the main topic.
The three very effective modes of gene transfer Transformation, Transduction and Transfection observed in bacteria fascinated the scientist leading to the development of molecular cloning. The basic principle applied in molecular cloning is transfer of desired gene from donor to a selected recipient for various applications in the field of medicine, research, gene therapy with an ultimate aim of beneficial to the mankind.

Transformation: Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. This single stranded DNA mingles with the host genetic material successfully.

The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation. In the chemical mediated gene transfer, the cold conditioned cells in calcium chloride solution are exposed to sudden heat which increases the permeability of the cell membrane allowing the foreign DNA. The electroporation method as the name indicates, pores are made in the cell by exposing it to suitable electric field, allowing the entry of the DNA. The opened up portions of the cell are sealed by the ability of the cell to repair.

Transduction: In transduction, a media like virus is required between two bacterial cells in transferring genes from one cell to the other. Researchers used virus as a tool to introduce foreign DNA from the selected species to the target organism. Transduction mode of gene transfer follows either a lysogenic phase or lytic phase. In the lysogenic phase, the viral (phage) DNA once joining the bacterial DNA through transduction stays dormant in the following generations. The induction of lysogenic cycle by an external factor like UV light results in lytic phase. In lytic phase, the viral or phage DNA exists a s a separate entity in the host cell and the host cell replicates viral DNA mistaking it for its own DNA.As a result many phages are produced within the host cell and when the number exceeds it causes the lysis of the host cell and the phages exits and infects other cells. As this process involves existence of both the genome of the phage and the genome of the bacteria in the same cell, it may result in exchange of some genes between the two DNA. As a result, the newly developed phage leaving the cell may carry a bacterial gene and transfer it to the other cell it infects. Also some of the phage genes may be present in the host cell. There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.

Transfection: One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. The significance of creating pores and introducing the DNA into the host mammalian cell contributed to different methods in transfection. Chemical mediated transfection involves use of either calcium phosphate or cationic polymers or liposomes. Electroporation, sonoporation, impalefection, optical transfection, hydro dynamic delivery are some of the non chemical based gene transfer. Particle based transfection uses gene gun technique where a nanoparticle is used to transfer the DNA to host cell or by another method called as magnetofection. Nucleofection and use of heat shock are the other evolved methods for successful transfection.
 
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#2
This is post no. 2 under the main topic.
Gene transfer is a technique to stably and efficiently introduce functional genes (that are usually cloned) into the target cells. Genes are the fundamental hereditary units of all the life forms. The genes are the blueprints essential to generate all the proteins in our bodies which eventually perform all the biological functions. Therefore, when a gene is efficiently introduced into a target cell of the host, the protein which is encoded by that gene is produced.

Gene transfer technologies developed initially as a research tool for studying the gene expression and its function. On the other hand, as novel gene transfer technologies developed and older technologies were sophisticated, its potential applications have expanded significantly. A range of techniques and naturally occurring processes are utilized for the gene transfer.

Chemical Methods:
DEAE-dextran (Diethylaminoethyl-dextran)- It is polycationic compound and is derived from dextran (a polymer of carbohydrate). DEAE-dextran is capable of binding to the anionic phosphodiester backbone of the deoxyribo nucleic acid (DNA) due to its positive charge. The resulting complex retains an overall cationic charge and is capable of binding to the surfaces of negatively charged cell membrane. Consequently, the complex is then taken up by the cell most probably by the process of endocytosis. The advantages of the DEAE-dextran transfection technique include its easiness, reproducibility, and lower cost.

Calcium Phosphate - Co-precipitation of Calcium phosphate is one of the most famous and broadly utilized techniques for transfection of DNA from the time when it was initially introduced by Van Der Ebin and Graham during early years of 1970’s. This method involves mixing the nucleic acid (DNA) with calcium chloride, and then cautiously adding this mixture to a saline solution with phosphate buffer accompanied by incubating the mixture at room temperature. This produces a precipitate with DNA, which is then spread onto cultured cells. The precipitate is then taken up by the cells by means of phagocytosis or endocytosis. The chief advantages of the calcium phosphate technique are that it is simple, applicable to a wide range of cell types and can be accomplished at lower cost.
 
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#3
This is post no. 3 under the main topic.
Lipofection- It is the most common and generally utilized gene transfer technique in the recent years. Transfection lipids (cationic) are made up of a positively charged head group (for instance amine), a flexible linker group (ether or ester) and 2 or more hydrophobic tail groups. The combined DNA and cationic lipids act instantaneously to form structures called as lipoplexes that are more complex in structure than the simple liposomes. When lipoplexes are prepared under suitable conditions, they sustain an overall positive charge, which enables them to effectively bind to negatively charged cell surfaces and enter the cell by means of endocytosis. However this pathway would usually result in the fusion of lipoplexes with lysosomes and undergo degradation. This problem is overcome by utilizing the neutral helper lipids, for instance dioleoylethanolamine (DOPE), which are generally included along with the cationic lipid. This allows entrapped DNA to escape the endosomes, reach the nucleus and get access to the cell’s transcriptional machinery.

Polymers – In the current era, a diversity of organic polymers are being used to carry out transfection. One of the most well accepted polymer is the, polyethylenimine (PEI). It is a polycationic organic macromolecule that has a high cationic charge density (also called as a proton sponge). It condenses the nucleic acid (DNA) into positively charged particle that interacts with the cell surfaces that are anionic in nature and gains entry into the cells by means of endocytosis. Dendrimers are another group of polymers that are composed of three-dimensional, branched structures known as dendrons. Among them, the polyamidoamine (PAMAM) family of dendrimers have proven to be a useful tool for transfection. Since the sphere-shaped polycationic dendrimers are alike in proportion and shape to the histone clusters, they can compact the DNA to a small size and facilitate its entry into cells.

Targeting Proteins & Peptides - A range of protein and peptide sequences have been utilized to target, enhance or mediate delivery of nucleic acids in a large variety of applications. Such proteins and peptides are regularly utilized along with cationic lipids (example integrin-targeting peptide, Fusogenic peptides such as GALA, N-terminal peptide of influenza hemagglutinin HA2 subunit). The key benefits of utilizing targeting proteins and peptides are that both will enhance the transfection efficiencies and provide targeted delivery.
 
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#4
This is post no. 4 under the main topic.
Conjugation
Another way of transfer of bacterial DNA between different cells is conjugation. This requires a direct cell to cell contact. The conjugation process was first described by Joshua Lederberg and Edward Tatum in 1946 after their discovery of F factor or episome of Escherichia coli cells. The F factor is very well studied conjugation system. Apart from the F factor there is numerous conjugation systems present within several bacterial systems. Often these conjugation systems carry antibiotic resistant genes and they are referred as R factor. Both F and R factors have machineries enabling them to auto-replicate thus get inherited by the daughter cells during binary fission. But additionally the conjugation process helps these plasmids to be transferred between the donor cells to a recipient one.
The bacterial conjugation involves the following steps: formation of pair after mating, synthesis of the conjugal DNA, transfer of DNA followed by maturation. The mating process is facilitated by a long structure formed between the two cells known as F or sex pilus. Though around twenty genes are responsible for a successful F pilus formation, it is mainly composed of one protein called pilin. F pilus retracts itself into the donor cell by removing the pilin protein monomers from the pilus base, thereby bringing the cells together in close proximity.
A specialized DNA replication is started after the formation of the stable mating pair. A single stranded copy of the F factor DNA is synthesized (unlike normal replication, giving rise to double stranded DNA) which is transferred into the recipient cell. Inside, the recipient cell the single stranded DNA is made double stranded thereby forming a matured double stranded plasmid. The mating pair gets broken at the end of the conjugation process with both the donor and recipient cell carrying one copy of the F factor. The daughter cells of the recipient from now on will inherit the F factor.
An F factor often contributes to the transferring of chromosomal DNA as well along with F factor genes. This happens when F factor gets integrated in chromosomal DNA and then it gets replicated along with the chromosomal DNA. Thus the daughter cell inherits it as a part of the chromosomal DNA. During further conjugation often a small part of the chromosomal DNA adjacent to the F factor also gets transferred to the recipient cell along with the F factor. In addition the F factor has the ability to transfer chromosomal DNA from the donor to the recipient cell along with itself. Since these DNA sequences encode bacterial genes, there is a possibility of recombination with the same genes present in the recipient DNA. These types of donor cells having an integrated copy of the F factor are called High frequency of recombination or Hfr strains.
 
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#5
This is post no. 5 under the main topic.
The opening post in this thread mentions the use of nanoparticles in gene transfection. Gene therapy depends on safe and efficient gene transfer and nanoparticles are potentially useful vectors in this regard. Up until now, the vectors most recently used in gene therapy were viral-based. However, there are various issues associated with viral vectors, such as host immune responses, residual pathogenicity and limited cargo capacities of, for example, adeno-associated viruses. Nanoparticle-based approaches therefore under intense study as targeted delivery vehicles for RNA and DNA genetic drugs.

In one study in the journal Nature Materials, silver nanoparticles were developed in which a phenomenon called plasmonics was exploited. This entails resonance of nanostructured materials including gold and silver in light such that their electromagnetic fields are concentrated near the surface. This facilitates detection of the nanoprobes as it allows enhancement of the fluorescence of dyes used in their labelling. An etching technique was used to facilitate breakdown of non-internalised and potentially toxic nanoparticles for elimination. The spherical nanoparticles were also coated with a peptide that allowed them to be targeted to tumour cells, sparing healthy cells. The spherical nanoparticles were readily internalised in the tumour cells; use of these types of nanoparticles would therefore overcome the inability of RNA and DNA genetic drugs to penetrate cell membranes. The intense fluorescence of the plasmonic nanoparticles also meant that internalisation was readily detectable, while the etching technology meant that excess nanoparticles could be broken down and expelled by the kidneys.
Another potential application for gene delivery via nanoparticles would be in the area of animal genetics and breeding. One recent study described use of spherical, magnetic Fe[sub]3[/sub]O[sub]4[/sub] nanoparticles for delivery of genes expressed on DNA plasmids into the nuclei of porcine somatic cells. The nanoparticle surfaces were modified using the polycationic polyethylenimine, after which the nanoparticles showed strong binding affinity for DNA plasmids expressing the genes encoding a green (DNAGFP) or red (DNADsRed) fluorescent protein. The weight ratios of the genes to the nanoparticles could be manipulated to ensure complete binding of the DNA by the nanoparticles, even for DNA of several hundred nanometers in length. Stable and efficient co-expression of the green and red genes could be achieved in porcine kidney cells by magnetofection. The study suggested the great potential of magnetic nanoparticles for gene delivery in animal genetics and breeding studies.

References:

Friman, T., Pang, H.-B., Pallaoro, A., de Mendoza, T. H., Willmore, A.-M. A., Kotamraju, V. R., Mann, A. P., She, Z.-G., Sugahara, K. N., Reich, N. O., Teesalu, T., and Ruoslahti, E. (2014). Etchable plasmonic nanoparticle probes to image and quantify cellular internalization. Nature Materials (8 June 2014), doi:10.1038/nmat3982

Wang Y, Cui H, Li K, Sun C, Du W, Cui J, Zhao X, Chen W. A magnetic nanoparticle-based multiple-gene delivery system for transfection of porcine kidney cells. PLoS One. 2014 Jul 21;9(7):e102886. doi: 10.1371/journal.pone.0102886
 
 
 
 
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