The genes are made up of DNA and they produce their phenotypic effects transcription and translation. Copies of DNA are produced through semi conservative replication. The high fidelity replication of DNA ensures transmission of genes from parents to progeny without any change; this is reason for stability of genetically controlled phenotypes overt generations. The mutations are ultimate source of all the heritable variation observed in living beings. The natural processes of gene transfer vary preferable in their range and specificity. In general, (1) they are rather imprecise, which makes the recovery of desired gene combination dependent on efficient screening and selection, (2) their range in terms of the species involved is rather restricted depending on sexual compatibility (sexual reproduction), and virus host range (transduction).
One would like to reproduce such and even more distant gene transfer in a controlled manner and at a high enough rates to be of practical application. This is achieved by the technique of recombinant DNA technology, which consists of isolation of multiple copies of a desired gene and then transferring this gene in to suitable organism (called host) to achieve one of the following two broad objectives: (1) large scale production of the protein encode by gene, and (2) expression of gene leading to the development of a desired phenotype in the organism.
The pure copied of desired gene can be obtained in the following three ways. (1) Gene Cloning is the basic strategy that must be used to obtain the first ever preparation of all the genes. (2) Once the information on the sequence of the gene becomes available, Polymerase Chain Reaction (PCR) can be used to prepare copies of the gene much more easily, rapidly and cheaply. (3) When the sequence of gene is known or even the amino acid sequence of its protein product is known, the gene could be synthesized either chemically or by combining PCR with chemical synthesis. The chemical synthesis of a gene provides an opportunity to affect large scale modification in the base sequence of the gene.
"A recombinant DNA molecule is produced by joining two or more DNA segments usually originated from different organisms" or a recombinant DNA molecule is a vector (plasmid, phage or virus) into which the desired DNA fragment has been inserted to enable its cloning in an appropriate host. This is achieved by using specific enzymes for cutting DNA (restriction enzymes) into suitable fragments and then for joining together the appropriate fragments (ligation). In this manner, a recombinant DNA molecule may be produced, which contains a gene from one organism joined to regulatory sequences from another organisms; such a gene is called chimeric gene.
Recombinant DNA molecules are procured with one of the following three objectives (1) to obtain a large number of copies of specific DNA fragments, (2) to recover large quantities of the protein procured by concerned gene, (3) to integrate the gene in the chromosome of a target organism where it expresses itself. All these steps concerned with piecing together DNA segments of reserve origin and placing them into a suitable vector together constitute recombinant DNA technology.
The vector containing DNA segments to be cloned, called DNA inserts, are then introduced into a suitable organisms, usually a bacterium (host) while the process is called transformation. The transferred host cells are selected and cloned. The recombinant DNA present in such clones can replicate either in synchrony or independent of host cells; the gene present in the vector may or may not express itself by directing the synthesis of concerned polypeptide. The step concerned with transformation of suitable host with recombinant DNA, and cloning of the transformed cells is called DNA cloning or gene cloning. DNA or gene cloning is taken to include both the development DNA as well as their cloning in a suitable host.
The entire procedure of gene cloning or recombinant DNA technology may be classified into the following five steps for the convenience in description and on the basis of the chief activity performed.
Cloning: Cloning involves the removal of the nucleus from the cell and its placed in an unfertilized egg cell whose nucleus has either been deactivated or removed. In simple terms, cloning consists of trypsinization of a monolayer culture to prepare a cell suspension, and sending in Petri dishes or flasks. The culture vessels are incubated for1-3 weeks with a medium change over one week; by this time colonies will develop. Colonies may be isolated (1) directly from multiwall dishes by trypsinization of usually such well, which contain only one cell at the start, (2) isolation of clones from Petri dishes are done by using cloning ring, which are placed around desired colonies after the medium is poured. Alternatively, (3) the desired colony may be shielded and remaining colonies irradiated by a lethal dose. The protected colony is trypsinization, and the cell as are cloned in the same plate, the irradiated cells serving as a feeder layer.
1. Reproduction Cloning. After a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus.
2. Therapeutic cloning. The egg is placed into a Petri dish where it develops into embryonic stem cells, which have shown potentials for treating several diseases.
In February 1997, cloning became focus of media when Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheep, named Dolly, from the mammary glands of an adult female. The cloning of Dolly made it apparent to many that the techniques used to produce her could someday be used to clone human beings. This stirred a lot of controversy because of its ethical implications. The cloning is used to;
i. Obtain homogeneous cell line from heterogeneous cell cultures.
ii. To isolate biochemical mutants.
iii. To develop hybridoma clones.
iv. Cell strains with market chromosomes.
Cloning is generally applied to continuous cell lines, but often clones of these lines become considerably heterogeneous by the time they are sufficiently multiplied for use. Problem with finite cell line is that of line-span; by the time the clone is sufficiently multiplied, the cell may be approaching senescence.
Transgenic animal contains in its genome a gene or genes which introduced by one or other technique of transfection. The gene introduced by transfection is called transgene. In animals, transfection specifies the introduction of DNA segment, either naked or integrated with vector, into an animal cell. It is simply known as transformation in all other organisms. However, in case of animals, transformation has long been used to describe the change of normal, i.e., non-tumourous, cells in culture to tumor like cells. Transfection may be transient or stable/permanent. In transient transfection, the introduced genes are gradually lost from the daughter cells of transfected cells. But in case of permanent transfection, the introduction genes are retained and expressed in all the cells derived from the transfected cells. Since most of the animal vectors are unstable, i.e, gradually lost, in the extra chromosomal state, stable transfections are ordinarily due to integration of introduced genes into the cell genome.
The various objectives for which transgenic cells/animals are produced may be summarized into following six categories.
1. A majority of gene transfers aim at studies of promoter function, reporter gene expression, regulation of gene expression and function of transferred genes.
2. Genes have been transferred and expressed into cultural cell lines to obtain the proteins encoded by them.
3. Genetic modification of animals may be aimed at improving their milk, meat and wool etc. production.
4. Genes have been transferred into animals with a view to obtain a large scale production of the protein encoded by these genes in a milk, urine or blood of such animals; such animals are often called bioreactors and the approach is referred to as molecular farming or gene farming.
5. A special case of gene transfer aims at decreasing or eliminating the symptoms and consequent miseries of genetic diseases. In this approach, normal and functional genes are introduced at the place of defective gene in patient (gene therapy).
6. Finally, specific transgenic animal strains or lines are created to fulfill specialized experimental and biomedical needs. A good example of such animals are “knock out” mice strains in which specific genes have been replace or knocked out by their disrupted counterparts through a process of homologous recombination.
Vector is a circular DNA molecule, which can carry the foreign gene, when insert into it. The various animal vectors are based on one or the other virus, e.g., SV40 vectors, bovine papillamovirus vectors, retrovirus vectors, etc.
The different types of vectors used for gene transfers in animals:
|1. SV40 vectors (a) Early region replacement vectors (b) late region replacement vectors||Deprived from Replacement of large-T gene of SV40 Replacement of VPI, VP1, VP2, and VP3 gene of SV40, e.g.SVGT-5||Produce virions, which infect the host cells Transient gene expression and mammalian cells are hosts of SV40|
|2. Bovine Papilloma Virus (BPV) vectors||Transforming region+pBR322||Shuttle vector; often pBR322 sequence deleted prior to transfection|
|3. Retrovirus vectors||pBR322+retrovirus sequences||Shuttle vector; integrates as provirus into mammalian genome and used in gene therapy|
|4. Polymavirus vectors||Polymavirus origin+pBR322||Similar to SV40 vectors; mouse cells used as host|
|5. Vaccinia virus||DNA insert placed with the thymidine kinase gene of virus by a process of recombination||Promising a live vaccine; DNA insert in pathogen gene encoding an antigen|
|6. P element vectors||Drosophila transposable element; minimum of21 bp inverted repeats borders and the neighbouring regions,+E.coli vector e.g pUC8;DNA insert up to 40 kb placed within the two borders||Gene transfer in Drosophila; a helper P element is need to provide the transposes necessary for transposition or insertion of the ecombinant P vector into Drosophila genome|
|7. Bacculovirus vectors||Nuclear polyhedroma virus (npv) polyhedron gene replaced by DNA insert; e.g., AcNPV (Autographi -california Nuclear Poluhedroma Virus) and BmNPV (Bombyx mori Nuclear Polyhedroma Virus) vectors||Produce virions; expression vector for production of transgenic proteins silk worms larvae (BmNPV vectors) and in Spodoptera frugiperda larvae or cultured cells (Acnpv vectors)|
The most potent biotechnological approach is the transfer of specifically constructed gene assemblies through various transformation techniques with the help of genetic engineering. The plants obtained through genetic engineering contain a gene or genes usually from an unrelated organism; such genes are called transgene, and the plants containing transgenes are known as transgenic plants. The first transgenic plant war produced in 1983, when a tobacco line expressing kanamycin resistance was produced. The transgenic crop varieties resistant to herbicides, insects or viruses or expressing male sterility, delayed ripening or slow fruit softening were developed. ‘Flavr Savr’ tomato was the first transgenic variety to reach the market; fruits of this variety remain fresh for a prolonged period. In 196, the area under transgenic varieties was ˜3 million hectares, which had increased to over 34 million hectares by 1998(12-fold increase) in merely two years.
In case of plants, Agrobacterium plasmids Ti and Ri are the most commonly used as vectors. Agrobacterium tumefaciens has the Ti plasmid; while Agrobacterium rhizogenes has the Ri plasmid, these plasmids have similar general features and can be interchanged between the two bacterial species. Both the plasmids have a T-region, which contains genes for opine metabolism and phytohormone independence in nature, this region is transferred into the plant cells and is integrated into their genomes. They have another region, called vir region. The vir of a nopaline type Ti plasmid contains 8 operons (designated as virA, virB, virC, virD, virE, virF, virG, virH). This region mediates the transfer of T-DNA in to plant genomes, and hence is essential for virulence, that is production of crowngall/hairy root disease; therefore, it is called the virulence region or vir region. The genes of vir region are not transferred themselves; they only induce the transfer of T-DNA. Some enzymes (endonuclease) are essential for the excision transfer of the T-region into the plant cells. The genes to be transferred are placed within the T-DNA of these plasmids.
Some DNA viruses like caulimoviruses and Gemini viruses can be used as vectors for gene amplification since viruses multiply in plant cells. But the virus genomes are neither integrated into plant genomes nor are they generally transmitted through seed, hence these vectors cannot be used for stable gene transfers. The cauliflower mosaic virus (CaMV) has been extensively studied but it has found only limited use as a vector; Gemini viruses may be more promising. Intact or modified plant viruses can be readily introduced into plant cells using Agrobacterium Ti or Ri plasmid as a vector, the viral genome being placed within their T-DNA; this phenomenon is called agroinfection. Agroinfection is feasible both in monocots and dicots, and Agrobacterium rhizogenes seems to be more infective than Agrobacterium tumefaciens in the case of cereals.
The transgenic plants or genetically modified plants (GMP) have both basic and applied users, which are briefly summarized below.
1. They have proved to be extremely valuable tools in studies on plant molecular biology, regulations of gene action and identification of regulatory sequences.
2. The specific genes have been transferred into plants to improve their agronomic and other features. Gene conferring resistance to abiotic stresses, e.g., herbicide, has been transferred into crop plants, which enables the use of biodegradable herbicides like glyphosate in otherwise susceptible crops.
3. Genes for resistance to various biotic stresses have been engineered to generate transgenic plants resistance to insects, viruses, etc.
4. Several gene transfer have been aimed at improving the produce quality, e.g., protein or lipid quality of transgenic plants; the efforts have met with a variable degree of success.
5. Transgenic plants are aimed at producing novel biochemical like interferon, insulin, immunoglobulins or useful biopolymers like polyhydroxy-butyrate, which are not produced by normal plants. These compounds are extracted from plants and can be use as pharmaceutical or industrial substrates. Cultivation of transgenic plants for the recovery of pharmaceutical compounds (medicine) is popularly known as pharming.
6. Transgenic plants have been produced were fed on such a plant produce, they became immunized against the pathogen. Therefore, use of transgenic plants as vaccine for immunization against pathogens is fast emerging as an important objective.
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