Biotechnology is the use of living organisms to develop useful products. Biotechnology is often used in pharmaceutical manufacturing. Notable examples include the use of bacteria to produce things such as insulin or human growth hormone. Other examples include the use of transgenic pigs for the creation of hemoglobin in use of humans.
Amongst the earliest uses of biotechnology in pharmaceutical manufacturing is the use of recombinant DNA technology to modify Escherichia coli bacteria to produce human insulin, which was performed at Genentech in 1978. [1] Prior to the development of this technique, insulin was extracted from the pancreas glands of cattle, pigs, and other farm animals. While generally efficacious in the treatment of diabetes, animal-derived insulin is not indistinguishable from human insulin, and may therefore produce allergic reactions. [2] Genentech researchers produced artificial genes for each of the two protein chains that comprise the insulin molecule. The artificial genes were "then inserted... into plasmids... among a group of genes that" [1] are activated by lactose. Thus, the insulin-producing genes were also activated by lactose. The recombinant plasmids were inserted into Escherichia coli bacteria, which were "induced to produce 100,000 molecules of either chain A or chain B human insulin." [1] The two protein chains were then combined to produce insulin molecules.
Prior to the use of recombinant DNA technology to modify bacteria to produce human growth hormone, the hormone was manufactured by extraction from the pituitary glands of cadavers, as animal growth hormones have no therapeutic value in humans. Production of a single year's supply of human growth hormone required up to fifty pituitary glands, [3] creating significant shortages of the hormone. [4] In 1979, scientists at Genentech produced human growth hormone by inserting DNA coding for human growth hormone into a plasmid that was implanted in Escherichia coli bacteria. The gene that was inserted into the plasmid was created by reverse transcription of the mRNA found in pituitary glands to complementary DNA. HaeIII, a type of restriction enzyme which acts at restriction sites "in the 3' noncoding region" [5] and at the 23rd codon in complementary DNA for human growth hormone, was used to produce "a DNA fragment of 551 base pairs which includes coding sequences for amino acids 24–191 of HGH." [5] Then "a chemically synthesized DNA 'adaptor' fragment containing an ATG initiation codon..." [5] was produced with the codons for the first through 23rd amino acids in human growth hormone. The "two DNA fragments... [were] combined to form a synthetic-natural 'hybrid' gene." [5] The use of entirely synthetic methods of DNA production to produce a gene that would be translated to human growth hormone in Escherichia coli would have been exceedingly laborious due to the significant length of the amino acid sequence in human growth hormone. However, if the cDNA reverse transcribed from the mRNA for human growth hormone were inserted directly into the plasmid inserted into the Escherichia coli, the bacteria would translate regions of the gene that are not translated in humans, thereby producing a "pre-hormone containing an extra 26 amino acids" [5] which might be difficult to remove.
Prior to the development and FDA approval of a means to produce human blood clotting factors using recombinant DNA technologies, human blood clotting factors were produced from donated blood that was inadequately screened for HIV. Thus, HIV infection posed a significant danger to patients with hemophilia who received human blood clotting factors:
Most reports indicate that 60 to 80 percent of patients with hemophilia who were exposed to factor VIII concentrates between 1979 and 1984 are seropositive for HIV by [the] Western blot assay. As of May 1988, more than 659 patients with hemophilia had AIDS... [6]
The first human blood clotting factor to be produced in significant quantities using recombinant DNA technology was Factor IX, which was produced using transgenic Chinese hamster ovary cells in 1986. [7] Lacking a map of the human genome, researchers obtained a known sequence of the RNA for Factor IX by examining the amino acids in Factor IX:
Microsequencing of highly purified... [Factor IX] yielded sufficient amino acid sequence to construct oligonucleotide probes. [8]
The known sequence of Factor IX RNA was then used to search for the gene coding for Factor IX in a library of the DNA found in the human liver, since it was known that blood clotting factors are produced by the human liver: [8]
A unique oligonucleotide... homologous to Factor IX mRNA... was synthesized and labeled... The resultant probe was used to screen a human liver double-stranded cDNA library... Complete two-stranded DNA sequences of the... [relevant] cDNA... contained all of the coding sequence COOH-terminal of the eleventh codon (11) and the entire 3'-untranslated sequence. [7]
This sequence of cDNA was used to find the remaining DNA sequences comprising the Factor IX gene by searching the DNA in the X chromosome:
A genomic library from a human XXXX chromosome was prepared... and screen[ed] with a Factor IX cDNA probe. Hybridizing recombinant phage were isolated, plaque-purified, and the DNA isolated. Restriction mapping, Southern analysis, and DNA sequencing permitted identification of five recombinant phage-containing inserts which, when overlapped at common sequences, coded the entire 35kb Factor IX gene. [9]
Plasmids containing the Factor IX gene, along with plasmids with a gene that codes for resistance to methotrexate, were inserted into Chinese hamster ovary cells via transfection. Transfection involves the insertion of DNA into a eukaryotic cell. Unlike the analogous process of transformation in bacteria, transfected DNA is not ordinarily integrated into the cell's genome, and is therefore not usually passed on to subsequent generations via cell division. Thus, in order to obtain a "stable" transfection, a gene which confers a significant survival advantage must also be transfected, causing the few cells that did integrate the transfected DNA into their genomes to increase their population as cells that did not integrate the DNA are eliminated. In the case of this study, "grow[th] in increasing concentrations of methotrexate" [10] promoted the survival of stably transfected cells, and diminished the survival of other cells.
The Chinese hamster ovary cells that were stably transfected produced significant quantities of Factor IX, which was shown to have substantial coagulant properties, though of a lesser degree than Factor IX produced from human blood:
The specific activity of the recombinant Factor IX was measured on the basis of direct measurement of the coagulant activity... The specific activity of recombinant Factor IX was 75 units/mg... compared to 150 units/mg measured for plasma-derived Factor IX... [11]
In 1992, the FDA approved Factor VIII produced using transgenic Chinese hamster ovary cells, the first such blood clotting factor produced using recombinant DNA technology to be approved. [12]
Recombinant DNA techniques have also been employed to create transgenic farm animals that can produce pharmaceutical products for use in humans. For instance, pigs that produce human hemoglobin have been created. While blood from such pigs could not be employed directly for transfusion to humans, the hemoglobin could be refined and employed to manufacture a blood substitute. [13]
Bristol-Myers Squibb manufactures paclitaxel using Penicillium raistrickii and plant cell fermentation (PCF).[ citation needed ]
Transgenic yeast are used to produce artemisinin, as well as a number of insulin analogs. [14]
Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes (EPEC, ETEC etc.) can cause serious food poisoning in their hosts, and are occasionally responsible for food contamination incidents that prompt product recalls. Most strains do not cause disease in humans and are part of the normal microbiota of the gut; such strains are harmless or even beneficial to humans (although these strains tend to be less studied than the pathogenic ones). For example, some strains of E. coli benefit their hosts by producing vitamin K2 or by preventing the colonization of the intestine by pathogenic bacteria. These mutually beneficial relationships between E. coli and humans are a type of mutualistic biological relationship — where both the humans and the E. coli are benefitting each other. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh faecal matter under aerobic conditions for three days, but its numbers decline slowly afterwards.
A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.
Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.
In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
Polly and Molly, two ewes, were the first mammals to have been successfully cloned from an adult somatic cell and to be transgenic animals at the same time. This is not to be confused with Dolly the Sheep, the first animal to be successfully cloned from an adult somatic cell where there wasn’t modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the Roslin Institute in Edinburgh, Scotland.
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.
The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.
Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.
Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemical engineering in order to focus on molecular level solutions to issues and problems in the life sciences related to the environment, agriculture, energy, industry, food production, biotechnology and medicine.
Ancestim is a recombinant methionyl human stem cell factor, branded by Amgen as StemGen. It was developed by Amgen and sold to Biovitrium, now Swedish Orphan Biovitrum, in December, 2008.
Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, a common lab bacterium and Agrobacterium tumefaciens, a bacterium used to insert the recombinant (customized) DNA into plants. Plant Transformation vectors contain three key elements;
In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors have an origin of replication, a multicloning site, and a selectable marker.
Genetically modified bacteria were the first organisms to be modified in the laboratory, due to their simple genetics. These organisms are now used for several purposes, and are particularly important in producing large amounts of pure human proteins for use in medicine.
Moroctocog alfa is a recombinant antihemophilic factor genetically engineered from Chinese hamster ovary (CHO) cell line. Chemically it is a glycoprotein. It is manufactured by Genetics Institute, Inc. and used to control and prevent hemorrhagic bleeding and prophylaxis associated with surgery or to reduce the number of spontaneous bleeding episodes in patients with hemophilia A. It is partially a recombinant coagulation factor VIII since it has an amino acid sequence which compares to the 90 + 80 kDa form of factor VIII (BDDrFVIII). It also has posttranslational modifications which are similar to those of the plasma-derived molecule. It can not prevent hemorrhagic bleeding associated with von Willebrand's disease since it is not a von Willebrand factor.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.
Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.
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