What is genetic engineering? The process involving genetic engineering.

 

Genetic engineering actually began with the discovery of DNA. This structure of nucleotides has been used to identify and manipulate genes of all living organisms, including humans. Genetic engineering is a process that alters an organism's genome through the introduction and/or removal of specific traits and there are several different methods that can be employed. The two most common methods involve either "recombinant DNA" – the transferring of genes from one organism into another – or by using chemicals and radiation to induce random mutations in the natural genomes.

Genes are what make up chromosomes responsible for encoding proteins as well as regulating those encoded protein's activities; they also determine many phenotypic characteristics such as or eye color. By adding, removing, or otherwise manipulating genes in an organism's genome it is possible to alter its phenotypic characteristics.

 

Gene Cloning

The first method of genetic engineering is known as gene cloning. This process takes advantage of the discovery that bacteria can be induced to take up foreign DNA by heat shock or electroporation. During this procedure, bacterial cultures are heated to 42 degrees Celsius which causes their membranes to open briefly. Plasmid vectors containing foreign DNA are then introduced into these cells and rapidly cool them preventing the membrane from closing again. Under these conditions bacteria lack certain proteins necessary for repairing damage done to their cellular walls; thus they integrate plasmids containing foreign DNA into their own genomes. Bacteria continue multiplying after this procedure, passing on the plasmids containing foreign genes to their progeny. This results in a large increase in the number of bacteria harboring plasmids with recombinant DNA.

Gene Splicing

The second method is known as gene splicing. Once again this process takes advantage of bacteria's ability to take up foreign DNA but this time it involves cutting out specific genes from its chromosome and pasting them into another organism's genome. Gene-splicing makes use of restriction enzymes which are naturally produced by various microorganisms that constitute these molecules of nucleotides capable or recognizing specific sequences in cellular DNA. They will then cleave that DNA at those points producing sticky ends. By treating these fragments with an enzyme known as DNA ligase it is possible to covalently link two different DNA fragments together. Scientists can exploit this process to cut out specified genes from an organism's genome and paste them into another species. This method of genetic engineering requires that restriction enzymes be chosen which will recognize specific nucleotide sequences within the target organism's genome as well as those in the foreign gene being inserted. Those enzymes are also capable of recognizing certain sites on the plasmid vectors containing these genes so they are able to insert the recombinant DNA into the proper location on its chromosome.

Hybridization

The first example of successful genetic engineering actually involved combining genes from very different organisms. This method, known as hybridization, was used by Herbert Boyer and Stanley Cohen in 1972 when they combined plasmids containing the genes for antibiotic resistance from the Gram negative bacterium, "Escherichia coli", with plasmids containing the genes responsible for creating tetracycline resistance in the Gram positive bacterium, "Streptomyces". The results of this experiment were bacteria that could produce both types of antibiotics.

More recently researchers have begun experimenting with gene therapy by using enzymes known as zinc-finger nucleases. These are proteins capable of binding to specific sequences on DNA and then inducing a double stranded break at that location by making use of their associated Zn+2 ions. If these breaks are repaired incorrectly they can lead to mutations resulting in many different diseases. Scientists are now able to design zinc-finger nucleases that recognize specific triplet sequences on the DNA molecule and induce mutations at those locations. Furthermore, they can create fusion proteins by fusing these enzymatic domains to transcription activator-like effector (TALE) proteins which are capable of binding to specific nucleotide triplets in genomic DNA. This allows scientists to target specific regions of the genome by utilizing the TALE protein's ability to bind its target sequence and recruit the zinc-finger domain responsible for inducing a breakage. Once this has been done, the defective gene may be replaced by a functional copy using homologous recombination.

 

Process Steps

The first step in creating a genetically modified organism is isolating a suitable piece of DNA from its source organism or construct a piece of DNA from scratch which will be put into the target organism.

The second step is to introduce that DNA into a suitable microbe where it can be incorporated and expressed by its target cell so as to achieve the desired trait.

 

Once you have isolated or created this recombinant DNA, the third step involves putting the foreign gene inside another more useful organism's cells. This can either be done in vivo via injection, exposure to chemical agents which permeate the cell membrane or even physical pressure via bombardment with microprojectiles coated in molecules capable of penetrating them or in vitro using vectors capable of transferring genes across species barriers (). Most often these are viruses such as retroviruses, adeniruses and most commonly bacterial plasmids. Vectors derived from viruses require intact viral genes for their replication and expression of the gene they are carrying, limiting their usefulness. However, bacterial plasmids can be used without any alterations to themselves provided they bear an origin of replication functional in the target cell. Plasmid vectors also make it possible to easily remove the foreign DNA after its purpose has been fulfilled inside the host cell.

 

The final step is assessing whether or not your experiment was successful by analyzing how your genetically modified organism functions compared with its unmodified counterpart. This commonly involves studying its rate of reproduction, lifespan and/or behavior; however, it may involve many other traits as well depending on what we’re seeking to achieve through creating a genetically modified organism.

 

Gene Insertion and Process

The gene insertion step is often one of the most difficult and time consuming processes involved in genetic engineering techniques. The process used to introduce foreign DNA into an organism's genome via plasmids is called transformation. This can be done by either chemical or physical means, although chemical agents are commonly preferred due to their low cost and tendency for high-efficiency transformation (). Chemical agents merely permeate through the outer membrane, making them much less efficient than physical methods which actually punch through the membrane allowing the entire plasmid to enter at once (). Vectors utilizing viruses can deliver their payload directly into host cells without any assistance at all if their appropriate receptor has been expressed on the cell surface. For example, adenovirus can bind to an exposed cellular adhesion molecule called E-cadherin and infect epithelial cells if it has been exposed due to physical damage or apoptosis, known as cell death.

 

The introduction of the exogenous DNA into the microbe's genome requires a gene that is capable of recognizing where in the genome foreign DNA should be inserted and at what location. This gene is often referred to as a recombination site, but may also involve other functional elements such as promoters which are necessary for transcription of specific genes. These sites are commonly required because these vectors are promiscuous by nature since they do not recognize any particular allele on their own unless they contain recognition sites for known integrases or nucleases. These enzymes cut and rejoin DNA strands at specific nucleotide sequences. For example, the enzyme integrase will join two recombination sites to create a new sequence that is recognized as foreign by the host cell (). A promoter is merely a stretch of DNA that must be near or upstream from where transcription of a particular gene should occur. Promoters are necessary for the expression of all genes including those located on exogenous DNA because they allow RNA polymerase to bind to their respective cistrons and initiate transcription.

 

The vectors used in genetic engineering require more than just genetic information; they also need some sort of regulator which can provide temporal and spatial control over when and where the vector's genes are expressed. This regulator is usually in the form of a plasmid or viral promoter that either does not require an external source of energy or can use one to enhance expression in target cells. The latter case is usually achieved by using weak promoters like the early and late promoters from SV40 virus.

 

Plasmids used for genetic engineering are generally derived from ones naturally occurring in bacteria, but they may also be constructed artificially. Artificial constructs are made by carefully stitching together pieces of DNA with specific nucleotide sequences which allow them to fit into the host cell's genome at two points that flank an exogenous gene inserted into its middle. These sequences are called loxP sites (pronounced "lox pee") and they allow recombination enzymes like Cre-recombinase to recognize them and excise the intervening DNA, removing it permanently from the host cell's genome.

The plasmid's remaining genetic information is then inserted into a site in the vector's genome called an attachment site. A vector with this kind of genetic material is known as a recombinant virus. A specific integration event chosen by the researcher is usually one that will not interfere or destroy any essential genes since viruses are often reliant on their host cells for replication. A targeted integration event can also be chosen when there are two weakly expressed yet nonessential genes near each other which can act like scaffolds for the incoming plasmid.

 

The process involving genetic engineering begins with creating or acquiring vectors that can be used for specific applications. Bacterial vectors include plasmids and naturally occurring viruses such as bacteriophages which vary in their levels of complexity. Viruses require the presence of a host cell for replication, but plasmids do not and are much easier to produce and handle.

 

The next step is choosing an appropriate site or sites within the vector's genome in which to insert one or more genes which code for a desired protein. The choice in integration site may involve basic scientific research in order to determine where a particular gene will result in the highest expression levels when introduced into that location. If there is no known function then it may also be advantageous to look at places with lots of codons that correspond directly with the desired gene's amino acids. This increases the chances that a proper translation will occur when transcription is initiated from the promoter in the plasmid.

 

In order to produce a recombinant virus, the chosen site(s) must be verified and then disabled with a small deletion or insertion so that they can no longer express their functional protein products without any disruption to normal cellular functions. The necessary pieces, called cassettes, for this process are usually made by PCR amplification of either cDNA or genomic DNA which have been extracted from cells growing exponentially in culture. In this way, it is possible to obtain full-length genes with intact regulatory regions. These cassettes can then be inserted at various sites within an expression vector containing suitable regulator sequences such as the human cytomegalovirus (hCMV) immediate early or late promoters, or in some cases the SV40 promoter.

 

After verifying that the modified vector behaves similarly to its parent strain when tested with cells growing in culture, it can be passed into target tissue by viral infection which usually occurs in vitro. The researchers then monitor for expression of the desired protein at various time intervals after infection by both detecting its mRNA levels and confirming functional activity through assays like chromogenic substrate assays which are commonly used to quantify reporter gene expression.

 

For example, a common strategy is to package an anti-sense RNA strand complementary to the targeted mRNA produced by cancerous cells into a recombinant virus containing a strong promoter sequence. After infection, the introduced vector's complementary RNA is used as a template for transcription into RNA that can then be translated into protein. The transcript results in a trans-dominant effect where expression of the targeted gene product is reduced or stopped completely. This can slow down tumor growth and may even lead to apoptosis which is programmed cell death.

 

In order to determine whether or not a specific recombinant virus has been successfully produced, the genetic material from both the host cell and vector must be extracted after infection so that each can be analyzed separately. When attempting to procure sufficient quantities of viral particles for further study, it is important to expose cells growing exponentially in culture to an agent that will promote viral production before harvesting. This allows researchers who are only interested in studying the viral components to skim off any cells that have been successfully infected and then induce further replication of these particles before they lyse and release, or spill their contents.

 

In prokaryotes such as E. coli, a bacteriophage may be engineered to express an antibiotic resistance gene so that progeny phages produced during infection will carry the new genetic material. This approach is frequently used in research involving bacteriophages because it tends to favor stable inheritance and integration into the bacterial chromosome which greatly increases its usefulness for future editing by recombinant DNA technology (2). Using plasmids for this purpose does not always result in stable integration and arbitrary site-directed mutation techniques would need to be used instead.

 

A similar approach can also be used to mutate multiple sites simultaneously which is achieved by co-infecting cells with two recombinant viruses that have different antibiotic resistance markers. The first marker is intended to replace one amino acid in the targeted gene sequence, whereas the second marker will replace another. After allowing these to multiply, both populations are analyzed using DNA sequencing techniques like Sanger sequencing or pyrosequencing which produce large amounts of data quickly and afford maximum accuracy. This process can be repeated several times until the desired mutation(s) are obtained. Both plasmids and bacteriophages can also be used to generate conditional lethal mutants for use in mammalian cell culture where they need only express their protein product for a finite amount of time to destroy the targeted cell.

Viruses have been used as gene transfer agents in vivo as well where they are modified so that they will only infect specific cells types and/or at a certain stage of development. The most common way to produce these is by using recombinant viruses that have been engineered to express ligands which bind to cell surface receptors located on target cells. One such receptor, epidermal growth factor receptor (EGFR) is present in most human carcinomas but not healthy tissue or during fetal development due to downregulation by transforming growth factors β (TGFβ). Thus, its natural ligand can be used with an EGF-expressing adenovirus to target and destroy cancer cells while leaving healthy cells alone. Vectors can also be engineered to express ligands which bind to surface receptors found in specific cell types or organ systems with the potential to treat a range of diseases from hemophilia A to lung cancer. In fact, trials using mesothelin-specific adenoviruses have been successful in decreasing both the size and number of tumors in patients who had previously failed all other treatments.