Gene splicing

Genetic engineering

Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes.Genetic Engineering is different from traditional breeding, where the organism's genes are manipulated indirectly; genetic engineering uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Genetic engineering techniques have found some successes in numerous applications. Some examples are in improving crop technology, the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster ovary cells, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research.

Since a protein sequence is specified by a segment of DNA called a gene, novel versions of that protein can be produced by changing the DNA sequence of the gene.

Engineering

  1. Isolation of the genes of interest
  2. Insertion of the genes into a transfer vector
  3. Transfer of the vector to the organism to be modified
  4. Transformation of the cells of the organism
  5. Separation of the genetically modified organism (GMO) from those that have not been successfully modified

Isolation is achieved by identifying the gene of interest that the scientist wishes to insert into the organism, usually using existing knowledge of the various functions of genes. DNA information can be obtained from cDNA or gDNA libraries, and amplified using PCR techniques. If necessary, i.e. for insertion of eukaryotic genomic DNA into prokaryotes, further modification may be carried out such as removal of introns or ligating prokaryotic promoters.

Insertion of a gene into a vector such as a plasmid can be done once the gene of interest is isolated. Other vectors can ones such as liposomes, or even direct insertion using gene guns. Restriction enzymes and ligases are of great use in this crucial step if it is being inserted into Werner Arber and Hamilton Smith received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases.

Once the vector is obtained, it can be used to transform the target organism. Depending on the vector used, it can be complex or simple. For example, using raw DNA with DNA guns is a fairly straightforward process but with low success rates, where the DNA is coated with molecules such as gold and fired directly into a cell. Other more complex methods, such as bacterial transformation or using viruses as vectors have higher success rates.

After transformation, the GMO can be isolated from those that have failed to take up the vector in various ways.

Genetic engineering and research

Although there has been a revolution in the biological sciences in the past twenty years, there is still a great deal that remains to be discovered. The completion of the sequencing of the human genome, as well as the genomes of most agriculturally and scientifically important animals and plants, has increased the possibilities of genetic research immeasurably. Expedient and inexpensive access to comprehensive genetic data has become a reality with billions of sequenced nucleotides already online and annotated.

  • Loss of function experiments, such as in a gene knockout experiment, in which an organism is engineered to lack the activity of one or more genes. This allows the experimenter to analyze the defects caused by this mutation, and can be considerably useful in unearthing the function of a gene. It is used especially frequently in developmental biology. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene which has been slightly altered such as to cripple its function. The construct is then taken up by embryonic stem cells, where the engineered copy of the gene replaces the organism's own gene. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. Another method, useful in organisms such as Drosophila (fruitfly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes.
  • Gain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently.

  • Tracking experiments, which seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting element such as Green Fluorescent Protein (GFP) that will allow easy visualization of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences which will serve as binding motifs to monoclonal antibodies.
  • Expression studies aim to discover where and when specific proteins are produced. In these experiments the DNA sequence before the DNA that codes for a protein, known as a gene's promoter is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as promoter bashing.

Human genetic engineering

Human genetic engineering can be used to treat genetic disease, but there is a difference between treating the disease in an individual and in changing the genome that gets passed down to that person's descendants (germ-line genetic engineering).

Human genetic engineering is already being used on a small scale to allow infertile women with genetic defects in their mitochondria to have children. Healthy human eggs from a second mother are used. The child produced this way has genetic information from two mothers and one father. The changes made are germ line changes and will likely be passed down from generation to generation, thus are a permanent change to the human genome.

Human genetic engineering has the potential to change human beings' appearance, adaptability, intelligence, character and behavior. It may potentially be used in creating more dramatic changes in humans. There are many unresolved ethical issues and concerns surrounding this technology, and it remains a controversial topic.

References

Reading list

Gene Splicing in Hollywood movies

  • The Fly A scientist is genetically spliced together with a fly during the teleportation process.
  • The Fly 2 A scientist's son swaps his mutant DNA with a healthy human's DNA; resulting in his cure of the "disease."

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