It is a genetic engineering term papers' purpose to examine just what cloning is, what it is for, and how it will affect our lives. However, biotechnology and genetic engineering involving agricultural products has become an area of increasing concern in the minds of population experts, trade negotiators, farmers and ranchers, seed companies and other suppliers, governmental officials and scientists.
Ultimately, questions related to the agricultural applications of biotechnology and genetic engineering are important to anyone who eats. With a world population expected to be over 8 billion early in the next century the ability to bring as much food to market for consumption becomes even more critical. With more land being taken up by urban sprawl leaving more marginal land to food production it is just that much more important to get as much production out of that land.
For many, the use of biotechnology and genetically engineered food or enhanced crops and livestock is the answer. There seems to be strong arguments on both sides of the question of genetically engineering crops and animals. I am for continuing the advancement of biogenetically engineered food because the positives far outweigh the negatives.
The genie of genetic engineering is out of the bottle and we cannot reverse science. We know how to do it, so it would seem best to do it for the best benefit for as many people as possible. Solving the problems associated with world hunger for the time being is a good use of that knowledge. While there may be some risks, there is no activity that is risk-free. The fear that is frequently expressed by the anti-genetic engineering side sometimes seems shrill. I wonder how much of modern technology and how many conveniences and scientific advances would have been dropped if those same neigh-sayers had been listened to then.
There always seems to be those who are fearful or suspicious of change and technology. While they are useful as a voice of caution, they should not be allowed to stop progress out of fear. What would happen if a genetically engineered virus or bacteria could escape the laboratory. This would be a virus which our immune systems have never encountered before.
Could it wipe out all human life? Several instances have been noted in which genetically engineered food items have resulted in widespread illness and even death. Genetics - One of the most exciting fields in genetics is genetic engineering. Genetic engineering when put another way could be described as designer people. Conservation Biology - Part of conservation biology incorporates genetic modification.
Somatic Cell Nuclear Transfer - Somatic Cell Nuclear Transfer research papers look into the laboratory technique used in genetics and developmental biology in which a viable embryo is created from a body cell and an egg cell. Ethics of Stem Cell - Ethics of Stem Cell research focus on the ethical considerations of medical health research. Breast Cancer Genetics - Breast Cancer Genetics research papers examine the leading type of cancer for women worldwide and look into the numerous risk factors.
Criminalizing Human Cloning - Criminalizing Human Cloning research papers examine genetic engineering and whether or not it should be a crime to clone people. Thus, E. The experiments conducted in and were crucial to the establishment of new genetics and genetic engineering.
Genome is considered long chains of nucleic acid that contains the information necessary to form an organism [ 2 ], consisting of small subunits called nucleic bases that are inheritable. Thus, the genome contains a complete set of features that are inheritable. The genome can be divided functionally into sets of base sequences, called genes. Each gene is responsible for coding a protein, and alternative forms called alleles.
A linear chain gene is named chromosome and each gene assumes a specific place, locus. Therefore, the modern view of genetics genome is a complete set of chromosomes for each individual. According to the central dogma Figure 1 , each gene sequence encodes another sequence of nitrogenous bases of single stranded RNA. As previously mentioned, each gene relates with expression of one protein and for that each codon the sequence of 3 nitrogenous bases of DNA represent only one amino acid, but each amino acid can be represented by more than one codon.
The DNA is considered as genetic material of bacteria, viruses and eukaryotic cells having a basic structure the nucleotide, which is formed by a nitrogenous base purine ring or pyrimidine , sugar and phosphate. In , Watson and Crick proposed that DNA is a double polynucleotide chain organized as a double helix. In this model, the double helix was linked by hydrogen bounding between nitrogenous bases.
The base is linked to the 1-position by a pentose glycosidic bond from N7 of pyrimidines or N9 of purine. The nuclear acid is named by the type of sugar. The chronological order of main events of genetic engineering and cloning are described above. Molecular biology and genetic were innovated in middle of 70 th decade the discover of restriction endonuclease by W Arber, D Nathans e H Smith that wan the Nobel price in Restriction endonuclease recognizes short sequences of duplex DNA as cleavage target and the enzyme cuts this point of DNA every time this target sequence occurs.
Analyzing restriction fragments is possible to generate a map of the original DNA molecule restriction map, a linear sequence of DNA separated in defined fragment size [ 1 , 2 ]. Restriction endonuclease are classified in types I, II and III by sequence specificity, nature of restriction and structural differences table 1. Types I and III have a restrict use in molecular biology and genetic engineering but the type II is largest used because it cleaves the DNA a specific recognition sequence, separate methylation, no additional energy requirement is necessar, high precision and do not match actions.
Type II restriction endonuclease are classified by the size of recognition sequence such as tetracutter, hexacutter or octacutter 4, 6 and 8 base paired respectively [ 3 ]. Restriction enzymes also could be classified as neoschizomers recognize the same sequence and isoschizomers recognize and cleave in the same location.
Properties of restriction endonucleases — Adapted from Satya, P[ 3 ] The nomenclature of restriction endonuclease is derivate from the species that it was isolated Ex. ECORI, from Escherichia coli Ry13 ; First two letters from enzyme name identify the species and the third identify the different strains from the same organism Table 2. The number classifies the different enzymes from the same organism and strains in chronological order of discover Ex. Restriction endonuclease cut the DNA in two different ways: blunt end two DNA strands are cleaved at the same position or sticky end the enzyme cut each DNA strand at different position, generally two until four nucleotides apart.
So in the sticky, DNA fragments have short single-stranded overhangs at each end. Same restriction endonuclease used in genetic engineering - Adapted from Brown, TA [ 4 ]. DNA ligase is a specific enzyme that is responsible to join DNA fragments spending and two blunt ends can be joined easily spending two ATPs molecules and this blunt end is very popular in genetic engineering.
Action of enzyme to catalyze the reaction is a random process that depends of vicinity of DNA fragments in solution. Joining DNA fragments with blunt ends is generally used to short oligonucleotides because concentration of free ends and enzyme are high, increasing the efficiency of process. Presence of sticky ends increase process efficiency because complementary ends come together by a random diffusion event in the solution and transient base pair might form between the two complementary strand.
This ligation is not very stable but may persist for enough time to join DNA fragments by DNA ligase catalysis and synthesis of phosphodiester bonds [ 4 ]. The greater efficiency of sticky-end ligation stimulated the creation of new methods, such linkers or adaptors. They are short double-strand molecules that cover the blunt-end and insert a recognition sequence for a restriction endonuclease to create a sticky-end. The linkers need to be digest by a restriction endonuclease to have a stick-end but the adaptor is a final sequence, digestion is not necessary and fragments can be direct joined by DNA ligase.
However, a single copy of recombinant DNA is not enough. Replication machinery of one organism generally is used to increase the number of copies. The DNA is inserted in the organism for a propagation or transfer. Generally, the vector has autonomic replication system that is independent of the cell cycle, increasing the number of copies.
To select recombinant cell some parameters need to be present: have restriction sites in which de exogenous DNA is inserted just one site for each restriction endonuclease and vector needs to have a marker gene multicloning sites one site for several restriction endonuclease makes the vector more useful [ 3 , 4 ]. The transgenic animal technology involves in first place, the isolation or artificially synthesis of a gene, which will be molecular manipulated and used for transformation leading to the transgenic production.
The need of knowledge involving this target gene can be overcome by its sequencing, conducting to the understanding of its structure. The main of this subject is briefly described the mechanisms involved in isolation, sequencing and synthesis of a gene.
The first gene isolation was reported in This method besides being resourceful work did not have general applicability. Now a day, several methods are in progress for isolation of a gene. A most traditional method used largely in research is the construction of a genomic or complementary DNA cDNA library. A genomic library represents the total DNA of a cell including the coding and non-coding sequences cloned on a vector and a cDNA library is a combination of cloned fragments from the mRNA inserts into a collection of host cells, creating in both cases, a portion of organism transcriptome.
To produce a genomic library after the extraction of genomic DNA, these molecules are digested into fragments of reasonable size by restrictions endonucleases and then inserted into a cloning vector generating a population of chimeric vector molecules. On the other hand, to create a cDNA library is necessary first, to produce a cDNA, which can be obtained from a mature mRNA isolate from a tissue or cells actively synthetizing proteins.
This cDNA are cloned into a bacterial plasmid, which is transformed into bacterial competent cells, amplified and selected. Once a genomic or a cDNA library is available, they can be used for the identification and isolation of a gene sequence. There are many commercial kits to create a genomic or cDNA library.
Normally, the genomic library is created with lambda or cosmid vectors while a cDNA library is produced with plasmid vectors more information see item 5. These kits usually try to improve the classical laborious techniques, enabling rapid construct of the libraries and ensuring generating of full-length clones.
The isolation of a gene using a genomic or cDNA library can be done by colony hybridization. In this technique the fragments containing a gene or parts of it can be identified by the use of DNA probes, which can be tagged or labeled with a molecular marker of either radioactive or fluorescent molecules. The commonly used markers are phosphorus 32 and digoxigenin, a non-radioactive, antibody-based marker. The DNA of bacteria carrying the chimeric vectors is fixed on the filter, which is hybridized with the labeled probe carrying a sequence related to the gene to be isolated.
The colonies carrying moderate to high similarity to the desired sequence are detected by visualizing the hybridized probe via autoradiography or other imaging techniques. In this way, the original chimeric vectors carrying the target gene sequence can be recovered from original colonies and used for advance researches.
If the library available were in the form of phage particles, instead of colonies are plaques that can be hybridized in the same way described above for colonies. This method of identification and isolation of genes are called plaques hybridization. To identify a gene related to a protein the inverse pathway from protein to DNA should be simulated. For start is necessary to have the protein product in a pure form.
To purify a protein several methods typically used are in a series of steps. Each step of protein purification usually results in some degree of product loss, so, an ideal strategy is one in which the highest level of purification is reached in the fewer steps.
The properties of the protein product like size, charge and solubility; determines the selection of which steps to use. These steps can be precipitation and differential solubilization; ultracentrifugation or chromatographic methods.
Thus, having the protein product is possible to produce antibodies probes for this protein by immunizing animals. This production require reliance upon animals immune system to levy responses that result in biosynthesis of antibodies against the inject molecule. Antigens must be prepared and delivered in a form and manner that maximizes production of a specific immune response by the animal. These antibodies probes can be used to precipitation of polysomes engaged in synthesizing the target protein leading to the achievement of the mRNA coded for this protein.
This method combined with immunoadsorbent techniques brings the possibility of application at less abundant proteins expression [ 6 ]. Thereby, to identify the specific cDNA clone for the target protein immunological and electrophoretic analysis methods are used, screening a complete or partial genomic library [ 10 ].
The basic concept of DNA sequencing is the mechanism involved in determining the order of nucleotides bases adenine, guanine, cytosine and thymine in a strand of DNA. Thus, at that moment, two methods of DNA sequencing were developed: one proposed by A.
Maxam and W. Gilbert, known as chemical method of DNA sequencing, and the other developed by F. Sanger, S. Nicklen and A. Coulson known as chain termination method. The chemical method of DNA sequencing consists in determines the nucleotide sequence of a terminally labeled DNA molecule by breaking it at adenosine, guanine, cytosine and thymine with chemical agents. Partial cleavage at each base produces a nested set of radioactive fragments extending from the labeled end to each of the positions of the base.
The autoradiograph of a gel produced from four different chemical cleavages, shows a pattern of bands from which the sequences are read directly [ 7 ]. The chain termination method depends on DNA replication and termination of replication at specific sequences. Both methods rely on four-lane high-resolution polyacrylamide gel electrophoresis to separate the labeled fragment and allow the base sequence to be read in a staggered ladder-like fashion.
Sanger sequencing was technically easier and faster, becoming the main basis of DNA sequencing, being modified and automated to aid large scale sequence procedure [ 3 , 8 , 9 ]. An automatic sequencing is an improvement of Sanger sequencing, through the use of different fluorescent dyes incorporated into DNA extension products primers or terminator. The use of different fluorophores in the four based A, C, G and T specific extension reactions means that all reactions can be loaded in a single lane.
For each base one color are used, emitting a different wavelength when excited. Throughout electrophoresis, the fluorescence signs are detected and recorded [ 10 , 11 ]. The classic electrophoresis methods used in automated sequencing are slab gel sequencing system or capillary sequence gel system, both described below. By this instrument, fluorescent-labeled fragments were loaded to the top of vertical gel and electric filed was applied, as the negatively charged DNA fragments migrated through the gel they were sized and fractionated by the polyacrylamide gel.
The fragments were automatically excited with a scanning argon laser and detected by a camera [ 12 ]. The loading of sequencing gels samples can be done manually or automatically. The automation consists in the use of a plexiglass block with wells in same distance from each other as the comb teeth cut in a porous membrane used as a comb for drawing samples by capillary action. The loading of samples automation achieve up to samples per gel [ 9 ].
Alternatively, the capillary sequence gel system instead of continuous polyacrylamide gel slabs, DNA is sent through a set of 96 capillary tubes filled with polymerized gel [ 3 , 9 ]. Solution phase DNA molecule are injected into the capillary either by pressure or electrokinetic injection and separated inside the capillary according to their size under high voltage conditions. The molecules are detected using UV light absorption or laser induced fluorescent detection at the end of the capillary [ 3 , 12 ].
The amplification of target DNA by PCR followed by direct sequencing of amplified DNA has emerged as a powerful strategy for rapid molecular genetics analysis bypassing the time consuming cloning steps and generating accurate DNA sequence information from small quantities of precious biological samples [ 14 ]. Some enzymes as Taq polymerase are thermostable and can be used in automated sequencing reactions such as cycle sequencing.
Others, such as Klenow polymerase and reverse transcriptase are thermal instable, being able to both direct sequencing by PCR products and cloned template, although cannot be used in cycle sequencing. Another enzyme, Sequenase, has also been used effectively in both radioactive and fluorescence cycle sequencing [ 8 ].
First, the PCR-amplified DNA is denatured to single strands, annealing the sequencing primer to complementary sequence on one of the template strands. Then, the annealed primer is extended by DNA polymerase by nucleotides, incorporating multiple radioactive labels into the newly synthesized DNA, under non-optimal reactions conditions, retaining the enzyme functionality low, for the synthesis of only short stretches DNA.
These methods generate high-intensity sequence ladders due to the advantage of automated cycling capability of thermal cyclers. First, the PCR-amplified DNA is denatured to single strands, and then it is annealed of a 32p-labeled sequencing primer. After, it is extended and chain-terminated by a thermostable DNA polymerase and denatured in the next sequencing cycle.
This step releases the template strand for another round of priming reactions while accumulates chain-terminated products in each cycle. These steps are repeated cycles to amplify the chain-terminated products in a linear fashion [ 14 ]. A DNA microarray technology brings the possibility of large scale sequence analyses by generating miniaturized arrays of densely packed oligonucleotide probes [ 9 , 16 ].
The word microarray has been derived from the Greek word mikro small and the French word arrayer arranged. This technology can be described as an ordered array of microscopic elements on a planar surface that allows the specific binding of genes or gene products [ 17 , 18 ]. The DNA sequencing by microarray uses a set of oligonucleotide probes to examine for complementary sequences on a target strand of DNA.
Briefly, after cleavage DNA segments are hybridized to the definitely arranged probes on a gene chip, the detection is made with a light driven. Then, to reconstruct the target DNA sequence, the hybridization pattern is used. To analyze the data and determinate the DNA sequence specific software are used [ 3 , 16 ]. The array elements react specifically with labeled mixtures, producing signals that reveal the identity and concentration of each labeled species in solution.
These attributes provide miniature biological assays that allow the exploration of any organism on a genomic scale [ 17 ]. The array technology has been widely used in functional genomics experiments designed to study the functions and interactions of genes within the context of the overall genome distinct plant and animal species. To sequence a DNA fragment by microarray a series of laboratory procedures are involved, from RNA extraction, reverse transcription and tagging fluorescent hybridization to the end, which invariably introduce different levels of additional variation data.
On the other hand, experiments with microarrays are still considerably expensive and laborious and, as a consequence, are generally conducted with relatively small sample sizes. Thus, the conducting tests on microarrays require careful experimental design and statistical analysis of the data [ 19 ].
The technique consists in embedded the samples to be analyzed in a crystalline structure of small organic compounds matrix and deposited on a conductive sample support. Then, the samples are irradiated with an ultraviolet UV laser with a wavelength of or nm.
The energy of the laser causes structural decomposition of the irradiated crystal and generates a particle cloud from which ions are extracted by an electric field. Following acceleration through the electric field, the ions drift through a field-free path and finally reach the detector. The results come from the calculation of ion masses by measuring their TOF, which is longer for larger molecules than for smaller ones.
Due to single-charged, nonfragmented ions are mostly produced, parent ion masses can be determined from the resulting spectrum without the need for complex data processing. The masses are accessible as numerical data for direct processing and subsequent analysis [ 20 ].
One of the four deoxynucleotides is replaced by an NTP. Fragments are generated by simple alkali backbone cleavage at the ribo-bases of the PCR products, generating oligonucleotide fragments each terminating with the ribonucleotide of the cycled primer extension reaction.
Differences between the unknown sample and a reference sequence are determined by changes in the results pattern [ 21 , 22 ]. Nowadays, with the advent of genome sequencing projects been accomplished, sequences of DNA can be obtained and compared through electronically databases, than physically from clone libraries described above.
The available databases include locus information, organism species, the whole gene sequence, the reference authors and the status of the sequencing. The gene synthesis methods had their main development during s and s. As DNA carries the genetic information of an organism, it could be viewed like a kind of information resource, enabling its reading sequencing, described above and writing synthesis.
The oligonucleotides synthesis can be done rapidly and in high yields with different kinds of methods. The gene synthesis, together with the knowledge of full genomes, molecular cloning, and protein expression profiles, improved the biotechnology field, making possible to explore the whole functionality of an entire complex organism. The gene synthesis machine is fully automated instrument, which synthesizes predetermined polynucleotide sequence.
The principle involved is based on a combination of organic chemistry and molecular biological techniques. Automatic gene machines, synthesize specific DNA sequences by programming the apparatus for the desired sequence. Briefly, the chosen sequence is entered in a keyboard and a microprocessor automatically opens the valve of nucleotide, chemical and solvent reservoir, controlling the whole process [ 15 ].
Containers of the four nucleotides A, T, C and G and reservoirs for reagent and solvent supports are connected with the synthesizer column. This column is packed with small silica beads, which provides support for assembly of DNA molecules. The desired sequence is synthesized on the silica beads which are later removed chemically [ 23 ]. Commercial services for gene synthesis are available from numerous companies worldwide. This gene synthesis method provides the possibility of creates entire genes without the need of a DNA template.
Temin and D. This enzyme, known as reverse transcriptase, are largely used in biotechnology research, and combined with the polymerase chain reaction create a methodology for DNA synthesis and amplification of the product. To use the mRNA as a template first is necessary purify this molecule of the cell, or tissue. Independently of efficiency the three kinds of mRNA isolation are available commercially, facilitating the lab work. Depending on the experiment, ligo dT , random hexanucleotides, or gene-specific antisense oligonucleotides can be used as primers for synthesis of first-strand cDNA [ 25 ].
The correctly native gene synthesis by this method depend on the fidelity of copying mRNA and also on the stability of DNA thus synthetized. Moreover, since mRNA of a gene does not have the complete transcript of the gene in vivo intronic regions are dismissed the synthesized gene will be smaller than the gene in vivo, but contain just the coding sequences, what could be a great advantage for research [ 9 ]. The gene synthesis by PCR, as described first for W.
Stemmer and coworkers were reported having four steps. First the olygos are synthetized, and then the gene is assembled, amplified and cloned. Since single-stranded ends of complementary DNA fragments are filled in during the gene assembly process, cycling with DNA polymerase results in the formation of increasingly larger DNA fragments until the full-length gene is obtained [ 26 ]. The classical method involves the use of oligonucleotides of 40nt long that overlap each other by 20nt.
The oligonucleotides are designed to cover the complete sequence of both strands, and the full-length molecule is generated progressively in a single reaction by overlap extension PCR, followed by amplification in a separate tube by PCR with two outer primers [ 27 ]. Variations of the classical approach were done, such as ligation of phosphorylated overlapping nucleotides, modified form of ligase chain reaction combinations with asymmetrical PCR and thermodynamically balanced inside out.
The most commonly synthesized genes range in size from to 1, bp although, much longer that genes made by connecting previously assembled fragments of fewer than 1, bp. In this size range it is necessary to test several candidate clones confirming the sequence of the cloned synthetic gene by automated sequencing methods [ 23 ].
The molecular cloning brings the possibility to isolate, analyze, synthetize and clone individual genes or segments of DNA, creating a recombinant DNA. After isolated and purified the DNA target sequence must be mounted on an appropriate carrier molecule, the cloning vector. A cloning vector is a small piece of DNA into which a foreign DNA is inserted for transfer or propagation in an organism, with the ability to self-replicate. The purpose of a vector is to allow efficient high-level expression of cloned genes or still, the need to increase the number of copies of a recombinant DNA [ 28 ].
The in vitro manipulation like, purification and transfer to a target cell, of a single copy is not possible. Thereby the recombinant construct should be propagated to increase the copy number. A convenient way to copy such fragments is to use the replication machinery of an organism, inserting the donor DNA in a cloning vector [ 29 ]. The essence of molecular cloning is to use restriction nucleases to cut DNA molecules in a starting DNA population the target DNA into pieces of manageable size, then attach them to a replicon any sequence capable of independent DNA replication and transfer the resulting hybrid molecules recombinant DNA into a suitable host cell which is then allowed to proliferate by cell division.
Because the replicon can replicate inside the cell often to high copy numbers so does the attached target DNA, resulting in a form of cell-based DNA amplification [ 11 ]. In principle, any molecule of DNA that can replicate itself inside a cell system could work as a cloning vector, but many factors as, small sizes, mobility between cells, easy production and detection mechanism should be considered [ 28 ].
The type of host cells used in a particular application will depend mainly on the purpose of the cloning procedure. Host cells exploited are modified bacterial, fungal cells e. Yeast , or still virus, being the bacterial system e. The vector may have an origin of replication that originates from either a natural extrachromosomal replicon or, in some cases, a chromosomal replicon [ 11 ].
Besides the structure the vectors should contain a sequence that make possible to select the recombinant cells, like a marker gene and in third place they should contain restriction sites into which the DNA can be inserted [ 29 ]. The types of cloning vectors are plasmids, phages, cosmids, phagemids, artificial chromosomes, viral vector and transposons. Each of them will be briefly describe in this section. Plasmids are small circular double-stranded DNA molecules, which exist in the cell as extrachromosomal units.
In a cell, they have the ability for self-replicating, and copy numbers maintenance. Due to their capacity of copy numbers they can be classified as: single copy plasmids or multicopy plasmids. The single copy plasmids are maintained as one plasmid DNA per cell, instead the multicopy plasmids that are maintained as copies per cell. Another kind of plasmids consists in ones that are under relaxed replication control, allowing their accumulation in numbers up to copies per cell, being the used ones as cloning vectors [ 15 ].
The plasmids vectors are designed to work in bacteria cells. An important property in these vectors is the detection of the same in the host cells. Usually, the detection mechanisms are done through antibiotic resistance. The host cell strain chosen is sensitive to a particular antibiotic and the plasmid is designed to contain a gene conferring resistance to this antibiotic. According to P. Gupta [ 15 ], there were three phases of plasmid development cloning vectors.
The first included the plasmids pSC, ColE1 and pCR1, which are naturally occurring plasmids, and not suitable for efficient cloning, since plasmid can transfer the gene through bacterial conjugation or can be integrated in the bacterial genome having no accessible detection system.
Other disadvantage lies on having no more than two restriction sites for cloning. The drawbacks of naturally occurring plasmids were overlapped by pBR and pBR The size reduction brought the pBR, which was largely used for many years. The second phase relies on reducing the plasmids sizes, because the transformation efficiency and vector size have a proportional inverse relation.
This plasmid vectors incorporate the selection mechanism of antibiotic resistance described above. Nowadays, there are a lot of plasmids commercially available that can be purchased depending on the application needs. A bacteriophage lambda is a bacterial virus that infects E.
Its utility as a cloning vector depends on the fact that not all of the lambda genome is essential for its function [ 1 ]. The lambda genome has the left-hand region with essential genes for the structural proteins and the right-hand region has genes for replication and lysis, while the middle region has the genes for integration and recombination, which are non-essentials.
There are two possible types of lambda vectors: the insertion vector and the replacement vector. The insertion vector has only a single recognition site for one or more restriction enzymes, enabling the DNA fragment to be inserted into the lambda genome. The lambda particle integrates DNA molecules between 37 and 52kb, and to adapt longer inserts is necessary to remove some of lambda genome. The region for replacement is the middle one where, more 23 kb of foreign DNA can be inserted.
This vector is known as replacement vector [ 28 ]. The replacement vector cannot be integrated into the host cells chromosome being necessary to use a helper phage to provide integration and recombination functions. On the other hand, this vector has two restriction sites, having a whole section of phage genome being replaced during cloning [ 1 ]. M13 is filamentous bacteriophages that infect specific E.
Your attractive as a cloning vector consists in its genomes contain the desirable size for a potential vector less than 10kb ; does not kill the host when progeny virus particles are released and thus, is easily prepared from an infected E. Besides, M13 is used as cloning vector to make single stranded DNA for sequencing and mutagenesis approaches. The M13 genome is a single-stranded DNA molecule with bp in length. This bacteriophage only infects bacteria carrying the F-pili fragile protein appendages found on conjugation-proficient cells , being male-specific.
When the DNA enters the cell, it is converted to a double-stranded molecule known as replicative form, which is a template for making about copies of the genome. At this point replication becomes asymmetric, and single-stranded copies of the genome are produced and extruded as M13 particles. The property of do not lyse the host cell brings a DNA resource, although growth and division are slower than in non-infected cells [ 1 , 11 , 28 ]. Cosmids are plasmid particles into which certain specific DNA sequences, namely those for cos sites, are inserted.
The goal of these vectors development is to cloning of large DNA fragments up to 47kb in length. The advantages consist of a highly efficient method of introducing the recombinant DNA and, a cloning capacity twofold greater than the best lambda replacement vectors. On the other hand, the gains of using cosmids instead of phage vectors are offset by losses in terms of ease to use and further processing of cloned sequences [ 1 ].
The methodology to use the cosmid cloning vectors consists in put together the cleaved vector and the target DNA for cloning, producing concatameric molecules. The concatameric molecules are usually generated by first linearizing the cosmid so that each end has cos site. Partial digestion leaves some site uncut and allows large segments of a genome to be isolated. These segments are mixed with the two halves of cosmid and joined using ligase. Thus, these molecules are packaged into phage heads by mixing with a packaging extract, becoming infectious.
Phagemids combine desirable features of both plasmids and bacteriophages. The construct consists of a plasmid with a segment of a filamentous bacteriophage, such as M13, having two different origins of replication: the plasmid and the phage origin. The selected phage sequences contain all the cis -acting elements required for DNA replication and assembly into phage particles [ 11 , 30 ].
These vectors allow successful cloning of inserts several kilobases. After E. These particles contain a mix of recombinant phagemids and helper phage. Vector pairs that have the phage origin in opposite directions are available, and as a result single stranded DNA representing of both DNA strands are produced. This mixed single strand DNA population can be used directly for DNA sequencing, if the primer for initiating DNA synthesis is designed to bind specifically to sequences of phagemid adjacent to the cloning site [ 11 , 30 ].
A bacterial artificial chromosome BAC is a single copy bacterial vector based on a functional fertility plasmid F-plasmid of E. BAC vectors are superior to other bacterial system, due to the F factor, which has genes regulating its own replication and controlling its copy number.
These regulatory genes are oriS and repE , mediating unidirectional replication and parA and parB , maintaining the copy number to one or two per cell. The cloning site is flanked by T7 and SP6 promoters for generating RNA probes for chromosome walking and for DNA sequencing of the inserted segment at the vector-insert junction. The CosN and loxP sites provides convenient generation of ends that can be used for restriction-site mapping to arrange the clones in an ordered way [ 31 ].
Besides the maintenance of large DNA inserts, BAC has structural stability in the host, high cloning efficiency and easy manipulation of cloned DNA, being largely utilized for construction of DNA libraries from complex genomes and subsequent rapid analysis of complex genome structure [ 31 ]. Transformed suitable E. The selection of recombined cells is done by hybridization procedures.
Viral vectors are commonly used to deliver genetic material into cells for gene therapy due to specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. This process can be performed inside a living organism in vivo or in cell culture in vitro , being frequently used to increase the frequency of cells expressing the transduced gene [ 32 ]. The first use of vector virus for cloning was based on simian virus 40 SV40 , a polyomavirus originated of rhesus macaque, being a potent DNA tumor virus infecting many types of mammal cells in culture.
The SV40 genome is 5. Due to packing limitations, cloning with SV40 involves replacing the existing genes with the foreign DNA [ 32 - 34 ]. Adenoviruses came to solve the size of insert drawback of SV40, enabling the cloning of DNA fragments up to 8kb. On the other hand, due to its larger genome, adenoviruses are difficult to handle. Expression can be transient and the in vivo transfection can be impaired due to immune response.
Papillomaviruses also have a high capacity for inserted DNA with the advantage of stable transformed cell line. Adeno-associated virus has this name because it is often found in cells that are simultaneously infected with adenovirus.
To complete the replication cycle the adeno-associated virus uses proteins already synthesized by adenovirus, which acts like a helper virus. Lack of helper virus made the genome of adeno-associated virus integrate to host DNA. The major advantage of this vector consist of a defined the insertion site, always in the same position, being important in researches that cloning gene needs rigorously check such as gene therapy. The herpesviruses include infections human viruses as herpes simplex virus HSV , most used like a vector.
The HSV is an enveloped double-stranded DNA, with kb, having advantages like larger foreign DNA carrying; high transduction efficiency and, potential to establish latency. Gene is stably integrated into the virus genome resulting in efficient replication and expression of biologically active molecules.
Many viruses kill their host cells by infection, so special artifices are needed if anything other than short-term transformation experiments is desirable. Bovine papillomavirus BPV , which causes warts on cattle, is particularly attractive because they have an unusual infection cycle in mouse cells taking the form of a multicopy plasmid with about molecules present per cell.
This infection does not bring the death of cell and, BPV molecules are passed to daughter cells during mitosis. The most used viral vectors are the retroviruses, infectious viruses that can integrate into transduced cells with high frequency, inserting the foreign DNA at random positions but, with great stability. They can be replicated-competent or replication-defective. Replication-competent viral vectors contain all necessary genes for virion synthesis, and continue to propagate themselves once infection occurs.
These vectors can integrate an inserted about 8—10 kb, limiting the introduction of many genomic sequences. This made replication-defective vectors the usual choice. These vectors had the coding regions replaced with other genes, or deleted. These viruses are capable of infecting their target cells but they fail to continue the typical lytic pathway that leads to cell lysis and death. The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme.
The vector, now called provirus, remains in the genome and is passed on to the progeny of the cell when it divides. The site of integration is unpredictable, which can pose a problem; therefore, the principal drawback of retrovirus vectors involves the requirement for cells to be actively dividing for transduction, being widely used in stem cells. Great examples to overcome this disadvantage are lentiviruses vectors. The lentivirus is a subset of retrovirus with the ability to integrate into host chromosomes and to infect non-dividing cells.
Lentivirus vector systems can include viruses of non-human origin feline immunodeficiency virus, equine infectious anemia virus as well as human viruses HIV. And for safety reasons lentiviral vectors never carry the genes required for their replication, preventing the occurrence of a wildtype-potentially infectious virus [ 32 - 34 ]. DNA transposons elements are natural genetic elements residing in the genome as repetitive sequences that move through a direct cut-and-paste mechanism.
This process is independent of previously recognized mechanisms for the integration of DNA molecules and occurs without need of DNA sequence homology. Thus, they can be used as tools from transgenesis to functional genomics and gene therapy.
Transposons are organized by terminal inverted repeats ITRs embracing a gene encoding transposase necessary for relocation. The development of transposable vectors is based on a plasmid system, with a helper plasmid expressing the transposase and a donor plasmid with terminal repeat sequences embracing the foreign gene [ 36 ]. The promoters are defined as cis -regulatory elements responsible for the control of transcriptional machinery and determination of its level and specificity, marking the point at which transcription of the gene should start, and regulating the transcription.
Promoters contain proximal elements, involved in the formation of the transcription complex; and, major elements that give cell specificity of protein expression [ 37 , 38 ]. For long term transgenic expression in vivo or tissue specific expression, the transcription of the foreign gene should be controlled for promoters, which in this case are inserted on cloning vectors [ 37 ].
Approaches requiring a high ubiquitous expression of the transgene can be accomplished with non-tissue specific promoters. These promoters are actives in almost all of cell types, ensuring the foreign gene expression in all organism tissues. Examples of these promoters are metallothionein gene promoter, EF1 gene promoter, CMV early gene promoter, human H2K gene promoter, 3-methylglutaryl CoA reductase gene promoters, and others. On the other hand, to restrict transgene expression to the target tissue the promoters used are tissue-specific.
These promoters can direct the transgene expression to lung, epithelia, liver albumin gene promoter , pancreas amylase promoter , muscles truncated muscle creatine kinase - MCK , neural cells synapsin 1 , mammary gland and cardiac cells troponin T promoter , and so on [ 38 ].
Promoters used in cloning vectors should be sufficiently short to be cloned in a gene transfer vector. Besides the use of tissue-specific promoters, another kinds of promoters are the inducible ones, which transcription can be selectively activated.
These promoters respond to specific transcriptional activators are: transcriptional activators regulated by small molecules; intracellular steroid hormone receptors; and, synthetic transcription factors in which dimerization is controlled by antibiotics. The promoters for transcriptional activators regulated by small molecules are based on the use of transcription factors that change their conformation upon binding one small chemical molecule e.
Tet repressor — TetR. Synthetic transcription factors in which dimerization are ones that in the presence of antibiotics tethers the transcriptional activation [ 37 , 38 ]. Transgenic animal technology and the ability to introduce functional genes into animals are powerful and dynamic tools of genetic engineering.
The genetic engineering field allows stable introduction of exogenous genetic information into any live organism, enhancing existing or, introducing entirely novel characteristics. The cloning technology is closely related with transgenic, being used as a tool for genetic engineering of an animal. Together these technologies can be used to dissect complex biological process, like in vivo study of gene function during development, organogenesis, aging, gene therapy, and epigenetics studies.
Besides, there are a lot of commercial applications like, model for human diseases, pharmaceutical biotechnologies development, and reproduction of a valuable animal.
The aim of this type of genetic modification is to cause organisms to exhibit new characteristics which are then passed to their offsprings Knoepffler Gene interaction is a type of chemical genetic modification in which several different genes are collaborated with an aim of producing a single phenotype or related traits.
This type of genetic engineering is commonly applied in the Mendelian experiments Hammond Gene mapping which is also known as a genome mapping is a type of chemical genetic engineering used to determine the sequence of certain features within the genome of a living organism. A genetic map is created using various markers on chromosome fragments. This type of genetic engineering is common in breeding experiments and pedigree analysis Anderson Molecular cloning is usually done at the molecular level which has been practiced in scientific research as a method of creation of many identical genes or cells for biomedical studies.
Each of the molecules and cells in the molecular cloning is similar to others. Molecular biologists have cloned DNA which is the molecular foundation of cells Knoepffler In molecular cloning, scientists copy and amplify fragments of DNA 2 which contain various genes into host cells which are normally bacteria. Molecular cloning is the basis for recombinant DNA technology which has been applied in the production of essential medicines. For example insulin was created using the molecular cloning and hence helped many diabetic patients.
In cellular cloning, cells which are derived from the body of a living organism are grown by scientists in a culture so that many copies of cells are created. The resultant cells are called cell lines and are similar to the original cells. This type of cloning procedure has also been applied in production of important medicines such as tissue plasminogen activator TPA which is used in treatment of heart attacks by dissolving clots Hammond Moreover, cellular cloning has been used in the insulin production.
It is important to note that in both molecular and cellular cloning, germ cells ova or sperms are not involved. Hence these types of cloning are not able to produce a baby. Embryo cloning is the cloning of animals and human beings which includes three categories: nuclear transfer, blastomere separation and blastocyst division.
Nuclear transfer is the cloning technique which was used in cloning Dolly the sheep by the Roslin Institute Hammond In nuclear transfer, a nucleus is removed from each of the blastomeres of a four to eight cell embryo and transferred or transplanted into the egg from which the genome was removed.
This is followed by artificial fusion of the membranes of the enucleated cell and the blastomere leading to the embryo development Cohen and Mary In blastomere separation, embryos are split immediately after fertilization. This is usually done at the two to eight cell stages where the blastomeres directed into production of multiple individual organisms have similar genetic composition.
Blastomere separation is commonly applied in the breeding of livestock Hammond Blastocyst separation which is also called twinning is a form of embryo cloning where embryos are split into two similar halves after they have sexually formed. This is followed by transfer of the two halves into the uterus leading to the development of identical twins Greene Nuclear transplantation was used in the Roslin Institute of Scotland to clone the first mammal from somatic cells of an adult.
Dolly the Ssheep was born in July 15 through nuclear somatic cell transfer in which a nucleus was transferred from a mammary cell of an adult sheep. The scientists induced fusion using electric pulses which enabled embryonic development of Dolly the sheep Cohen and Mary Therefore the success of cloning experiments in the Roslin Institute led to the increased interest of many scientists in the area of genetic engineering. Moreover, curiosity of many researches in genetic engineering has led to the need to genetically clone human beings.
This has led to diverse opinions on the ethics of artificially creating human clones as opposed to the natural methods of reproduction. Human cloning is simply defined as the creation of an indistinguishable copy of a human being through the use of modern genetic engineering technology.
Human cloning also refers to artificial creation of human beings. It is therefore different from the natural reproduction of human tissues Wilmut Human clones are thus created as a result of human cloning and they appear as identical twins. Artificial cloning of cells, molecules, plants and animals leads to creation of genetically similar copies without the involvement of sexual process. Therefore human cloning is the asexual replication of human beings and it is usually possible at any point of development.
In the modern biomedical research, human cells, genes, tissues and proteins are cloned with the aim of promoting human life Knoepffler As a result, this form of genetic engineering has raised several ethical issues which cause debates within the society. The two major types of human cloning are the reproductive and therapeutic cloning.
The creation of cloned human beings is a form of reproductive cloning while therapeutic cloning is the creation of human cells from an adult which are used in medicine Hammond There is also a theoretical possibility of human cloning referred to as replacement cloning which is a combination of reproductive and therapeutic cloning to replace damaged body cells as a way of recovery from a failing body which would be followed by partial or complete brain transplant Greene However, replacement cloning has not been possible but a lot of scientific research is currently focused in the area of genetic engineering.
This was a hybrid clone which was created from an egg of a cow and a cell from the leg of a man. The embryo which resulted would not be seen as human and thus was destroyed by the scientists after 12 days. The aim of this experiment was not reproductive cloning but rather it was aimed at therapeutic cloning Cohen and Mary The second attempt to clone a human being was in January when the Stemagen Corporation Laboratory in California created five mature embryos.
The scientists used genes from adult skin cells of a human being which were transferred to human ova. However the embryos were destroyed because the scientists said that it would be illegal and unethical if reproductive cloning was applied to create human beings Hammond Human cloning which applies the somatic cell nuclear transfer can be applied in creation embryos which are to be used in research. The creation of embryos for research purposes is a process called therapeutic or research cloning Greene The aim of this process is to harvest stem cells from human clones which are used to study the embryonic development of human beings which can be used as a basis of providing treatment for various diseases.
This application of cloning has enabled scientists and researchers to understand the cause of congenital diseases. As a result preventable measures have been taken to enable the protection of newborns from development of congenital deformities Hammond Human cloning can be used in assisted reproduction which helps many patients by making pregnancies which would not otherwise have happened to be possible.
The cloning technologies have been used in reproduction with minimal input of genomes from other parties. In vitro fertilization, donor sperm and ova in addition to surrogate motherhood have been made possible through artificial methods of assisted reproduction Wilmut There are circumstances when couples are not able to give birth especially in same sex marriages and when one of the parents is enable to provide the reproductive cells Hammond Therefore the application of cloning technology in assisted reproduction has assisted many couples in reproduction of human offsprings.
The cloning technology can be used to produce individuals with tissues which are immunologically similar to an existing being. This will assist in tissue donation Wilmut The transplantation of vital organs such as kidneys, liver heart has been a challenge in medical practice. This is due to incompatibility of human organs caused by differences in the genetic makeup among people. Many patients die as a result of failure to get a matching organ for the transplant Knoepffler This therefore justifies the use of cloning technology to produce the required donor organs and tissues.
However there are ethical and legal issues associated with the use of cloning technology in the production of donor tissues which have resulted into many divergent opinions on this issue. The cloning technology can be used to ensure healthy children. The current challenge in reproductive medicine is not to produce more embryos but to identify healthy ones and get them to grow in the womb.
Using genetic tests, doctors can now screen embryonic ells for hereditary diseases. In the not to distant future, prenatal tests may also help predict such common problems as obesity, depression and heart disease. The technological obstacles are formidable, and so are the cultural ones. Copies of humans are identical, but are the people the same?
Probably not. For a century scientists have been trying to figure out which factors play the most important role in the development of a human personality. Is it nature or nurture, heredity or environment? The best information so far has come from the study of identical twins reared apart.
Twins Jim Springer and Jim Lewis, separated at birth in , were reunited 39 years later in a study of twins at the University of Minnesota. Both had married and divorced women named Linda, married second wives named Betty and named their oldest sons James Allan and James Alan. Both drove the same model of blue Chevrolet, enjoyed woodworking, vacationed on the same Florida beach, and both had dogs named Toy.
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However, progress needs to be made on this important issue, especially for those genetically engineered species that are intended for life outside the research laboratory, where there may be less careful oversight of animal welfare. Consequently, limits to genetic engineering need to be established using the full breadth of public and expert opinion.
This highlights the importance for veterinarians, as animal health experts, to be involved in the discussion. Preserving intellectual property can breed a culture of confidentiality within the scientific community, which in turn limits data and animal sharing. Such limits to data and animal sharing may create situations in which there is unnecessary duplication of genetically engineered animal lines, thereby challenging the principle of Reduction.
Indeed, this was a concern that was identified in a recent workshop on the creation and use of genetically engineered animals in science It should be noted that no matter what the application of genetically engineered animals, there are restrictions on the methods of their disposal once they have been euthanized. The reason for this is to restrict the entry of genetically engineered animal carcasses into the natural ecosystem until the long-term effects and risks are better understood.
As genetically engineered animals begin to enter the commercial realm, it will become increasingly important for veterinarians to inform themselves about any special care and management required by these animals. As animal health professionals, veterinarians can also make important contributions to policy discussions related to the oversight of genetic engineering as it is applied to animals, and to regulatory proceedings for the commercial use of genetically engineered animals.
It is likely that public acceptance of genetically engineered animal products will be an important step in determining when and what types of genetically engineered animals will appear on the commercial market, especially those animals used for food production.
Veterinarians may also be called on to inform the public about genetic engineering techniques and any potential impacts to animal welfare and food safety. Consequently, for the discussion regarding genetically engineered animals to progress effectively, veterinarians need to be aware of the current context in which genetically engineered animals are created and used, and to be aware of the manner in which genetic engineering technology and the animals derived from it may be used in the future.
Genetic engineering techniques can be applied to a range of animal species, and although many genetically engineered animals are still in the research phase, there are a variety of intended applications for their use. Consequently, even if animal welfare can be satisfactorily safeguarded, intrinsic ethical concerns about the genetic engineering of animals may be cause enough to restrict certain types of genetically engineered animals from reaching their intended commercial application.
Given the complexity of views regarding genetic engineering, it is valuable to involve all stakeholders in discussions about the applications of this technology. Schuppli for her insight on how the issues discussed may affect veterinarians.
Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office gro. National Center for Biotechnology Information , U. Journal List Can Vet J v. Can Vet J. Elisabeth H. Ormandy , Julie Dale , and Gilly Griffin. Author information Copyright and License information Disclaimer. Address all correspondence to Dr. Griffin; e-mail: ac. This article has been cited by other articles in PMC.
Current context of genetically engineered animals Genetic engineering technology has numerous applications involving companion, wild, and farm animals, and animal models used in scientific research. Wild animals The primary application of genetic engineering to wild species involves cloning. Research animals Biomedical applications of genetically engineered animals are numerous, and include understanding of gene function, modeling of human disease to either understand disease mechanisms or to aid drug development, and xenotransplantation.
Ethical issues of genetic engineering Ethical issues, including concerns for animal welfare, can arise at all stages in the generation and life span of an individual genetically engineered animal. Concerns for animal welfare Invasiveness of procedures The generation of a new genetically engineered line of animals often involves the sacrifice of some animals and surgical procedures for example, vasectomy, surgical embryo transfer on others.
Unanticipated welfare concerns Little data has been collected on the net welfare impacts to genetically engineered animals or to those animals required for their creation, and genetic engineering techniques have been described as both unpredictable and inefficient Implications for veterinarians As genetically engineered animals begin to enter the commercial realm, it will become increasingly important for veterinarians to inform themselves about any special care and management required by these animals.
Footnotes Use of this article is limited to a single copy for personal study. References 1. Terrestrial Animal Health Code; p. The welfare implications of animal breeding and breeding technologies in commercial agriculture. Livestock Sci. Cloning Fact Sheet Available from www. Somatic cell nuclear transfer. West C. Economic and ethics in the genetic engineering of animals. Harvard J Law Technol. Cell biology: A cat cloned by nuclear transplantation. Dogs cloned from adult somatic cells.
Wildlife conservation and reproductive cloning. Laible G. Enhancing livestock through genetic engineering — Recent advances and future prospects. Comp Immunol Microb. Europeans and Biotechnology in Eurobarometer FDA Report August 25, Making recombinant proteins in animals — different systems, different applications. Trends Biotechnol. Wells DJ. Genetically modified animals and pharmacological research. Comparative and Veterinary Pharmacology. Berlin: Springer; Utilization of genetically altered animals in the pharmaceutical industry.
Toxicol Pathol. Logan JS, Sharma A. Potential use of genetically modified pigs as organ donors for transplantation into humans. Clin Exp Pharmacol P. Einsiedel EF, Ross H. Animal spare parts? A Canadian public consultation on xenotransplantation. Sci Eng Ethics. Macnaghten P. Refinement and reduction in production of genetically modified mice. Ormandy EH. The use of genetically-engineered animals in science. March 15, ; [accessed March16, ].
Worldwide trends in the use of animals in research: The contribution of genetically modified animal models. Sharing and archiving of genetically altered mice: Opportunities for reduction and refinement. Canadian Council on Animal Care. Trends in Animal Use. Gauthier C. Overview and analysis of animal use in North America. Verbeek JS. Scientific applications of transgenic mouse models.
Berlin: Springer-Verlag; Yoshiki A, Moriwaki K. Mouse phenome research: Implications of genetic background. Risks associated with genetic modification: An annotated bibliography of peer reviewed natural science publications. J Agr Enviro Ethic. Assessing the welfare of genetically altered mice.
Lab Anim. An improved zinc-finger nuclease technology architecture for highly specific genome editing. Nat Biotechnol. Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus. After Dolly — ethical limits to the use of biotechnology on farm animals.
Oritz G, Elizabeth S. Beyond welfare: Animal integrity, animal dignity, and genetic engineering. Ethic Enviro. Rollin BE. On telos and genetic-engineering. Animal Ethics Reader. London, UK: Routledge; Veerhoog H. Adults or adolescents can—through what Habermas calls a revisionary learning process p 62 —rid themselves of the effects that their parents' environmental decisions have produced on their minds, or alternatively, they can endorse their parents' environmental choices.
Such a picture of psychological development is flawed. Biologists and psychologists have shown that many environmental effects on psychological development are irreversible—and this applies in particular, but not exclusively, to environmental effects that occur in early childhood—and they have shown that many genetic effects on psychological development are reversible.
Thus, we can appeal to no such asymmetry to argue that people whose genome has been chosen are less responsible for their actions than those conceived through standard means. Various replies to arguments of this kind have been elaborated. Some authors have claimed that a person's false beliefs cannot provide moral grounds for restricting another person's freedom. I want instead to explore what I believe is a better even though not incompatible reply.
The costs of refusing to take responsibility are very high. As suggested by Habermas himself, the price to pay for the disavowal of responsibility for our actions is the exclusion from full participation in society. In a different but related context, Dennett 14 says something relevant:. No, we are not […] People want to be held accountable. The benefits that accrue to one who is a citizen in good standing in a free society are so widely appreciated that there is always a presumption in favor of inclusion.
Blame is the price we pay for credit, and we pay it gladly under most circumstances. We pay dearly, accepting punishment and public humiliation for a chance to get back in the game after we have been caught out in some transgression. They would not believe this because they would in general be aware of or they would learn very quickly about the disadvantages that such belief brings with it. After all, people conceived through standard means are sometimes tempted to disavow responsibility for some of their actions again, the socially undesirable ones and to claim that the responsibility for such actions falls on their parents and on their parents' educational choices.
Yet, we do not normally take this as a reason for prohibiting parents in general from choosing how to educate their children. We could start teaching people from an early age that some of a person's traits are the result of his or her parents trying—through genetic or environmental means—to raise the chances of those traits developing and that this does not at least not by itself in any way affect the moral status of this person.
And so on. Some forms of depression cannot be cured or avoided by making people aware of the fact that their being depressed is unjustified and irrational and that there are high costs attached to being depressed. In some cases, the process leading to the state of depression is or involves a reasoning process. In other cases, the depression is due merely to organic causes. In the latter cases, it is not possible to avoid the emergence of the depression by making the person realise that it would be irrational or disadvantageous for him or her to become depressed.
Given this, to see whether the analogy is correct, we need to focus only on those cases in which the depression results from a reasoning process. Consider the following situation: A person starts thinking about her life and after careful consideration she concludes perhaps correctly, perhaps incorrectly that she has reasons to believe that her life is going very badly.
She adopts the belief that her life is going very badly, this belief makes her depressed and the depression makes her very unlikely to revise the bleak view she has of her life. Once the depression kicks in, telling this person or even making her realise that her being depressed is unjustified and irrational has basically no effect on her depression. But as the depression is partly the result of a reasoning process, the depression could have been avoided by giving the person reasons to reject the adoption of the belief that her life was going very badly.
After all, many people refuse to believe that their life is going very badly even when they have good evidence for the truth of this belief and probably conscious or unconscious knowledge about the costs attached to being depressed has a role in this. This may be the case, even though, at the moment, we have no evidence for it. For example, they could choose genes that make their children severely mentally handicapped. Such parents would intentionally make their children disabled.
Arguably, their action would constitute an abuse and would have to be punished accordingly. But the remote possibility of such misuses does not by itself provide support for general restrictions on the reproductive use of genetic engineering and cloning. This is shown by the fact that parents can of course interfere with the cognitive and emotional development of their children through perverse and abusive environmental interventions, interventions that can result in their children becoming defective moral agents.
This can happen, for example, in the case of parents who interfere with the development of their children's brain by giving them powerful addictive drugs, by beating them violently or by keeping them as recluses for many years. We do not normally take the relatively rare occurrences of abuses of this sort as a reason to ban parents in general from choosing how to bring up their children.
Similarly, we should not take the remote possibility that some parents may misuse cloning and genetic engineering in the ways described as a reason to ban the reproductive use of these technologies. Buchanan et al 9 argue that reproductive genetic engineering can, in some circumstances, infringe a child's right to an open future and that this fact should seriously be taken into account when deciding whether to ban this reproductive technology. What is the right to an open future? It is not easy to extract a single definition from Feinberg's original article.
Buchanan et al suggest that the best way to make sense of Feinberg's notion is as follows:. The idea is that parents have a responsibility to help their children during their growth to adulthood to develop capacities for practical judgement and autonomous choice, and to develop as well at least a reasonable range of the skills and capacities necessary to provide them the choice of a reasonable array of different life plans available to members of their society.
Feinberg's original article is about parents' environmental rather than genetic choices. They argued that schooling beyond age 14 is not necessary for the Amish way of life and interferes with Amish children acquiring the skills and motivation needed to become an integrated member of the Amish community. The Wisconsin Supreme Court accepted their request. The child would not develop the skills necessary to pursue life projects different from those that can be pursued as a member of the Amish community.
According to Buchanan et al , 9 the principle that parents should not be allowed to make choices resulting in their children not having a reasonable array of life plans from which to choose should be applied to both environmental and genetic choices. Thus, a genetic intervention that makes a child particularly fit to pursue a career as, say, a pianist but unfit to pursue any other available career, would be illegitimate, especially in contemporary Western societies where a relatively large range of choices is usually available to most people.
Genetic interventions that make children fit for only a restricted range of ways of life violate the right to an open future and should thereby be banned. The ability to choose our own life plan is arguably one of the essential conditions of the good life. What does this ability require? People must have cognitive and emotional skills that make them able to a compare consciously or unconsciously different life plans, b select one among those life plans they are able to consider, c transform this choice into the intention to behave in accordance with the chosen plan and d transform this intention into behaviour that actually conforms to the chosen option.
Moreover, people must have skills that allow them to pursue different life plans with some definite chance of success, and they must be in a social context where these different life plans can actually be pursued. If people have skills that make them fit for one and only one particular and very specific life plan, they cannot be said to actually be able to choose their own life plan.
Similarly, if people live in a despotic society where they are allowed to pursue only one kind of career, they are obviously not free to choose their life plan. Parents inevitably exert an important influence on the array of life plans available to their children. In many cases, this influence results in children having a larger array of life plans from which to choose than they would otherwise have had.
In other cases, the influence results in children having a smaller array of life plans from which to choose than they would otherwise have had. Making someone fit for a particular life plan often but not always results in making the same person less fit for other life plans. Many parents make environmental choices aimed at increasing their children's chances of succeeding in the pursuit of more or less specific life plans.
By doing so, they often make their children less likely to succeed in the pursuit of other alternative life plans and, in this sense, they may reduce the range of life plans available to their children. Yet this sort of parental behaviour is in most cases considered legitimate, and some see it as an inevitable ingredient of being a good parent.
If the practice is legitimate in the case of most parental environmental choices, why should it not be legitimate in the case of most parental genetic choices? Parents are currently allowed to adopt relatively severe educational methods aimed at, for example, transforming their children into successful tennis players or into successful law school graduates. Given this, why should they not be allowed to use genetic methods to achieve similar results?. The discussion about the legitimacy of the Amish parents' request suggests that there is a moral limit to the extent to which parents can be allowed to reduce the array of life plans available to their children.
If some environmental choices eg, not sending a child to school reduce the range of life plans available to a child below a certain threshold, then those choices can be said to violate the child's autonomy and are thereby illegitimate.
Exactly the same applies to genetic choices. What is the threshold that parental choices—be they genetic or environmental—should never trespass? This is a difficult question and there is no room here for dealing with it properly. The correct answer to this question depends on many different factors.
Buchanan et al , 9 for example, argue that the answer depends, among other things, on what the correct theory of justice is. Despite the question being a difficult one, we can examine current practice for some suggestions about how to think about this issue. For example, we may notice that there exist relatively large disparities in the range of life plans available to different members of society.
Some of these disparities are due to unjust social arrangements, but arguably some of them are not. Rich people usually have more opportunities than poor people, and at least some of these differences in opportunity are usually not considered to be the result of unjust social arrangements. Yet it seems wrong to claim that, except perhaps in cases of extreme poverty, poor members of society violate their children's right to an open future when they decide to give birth to a child and not to give up their child for adoption to rich members of society.
Another consideration is that cloning and genetic engineering would probably not be used to reduce the array of life plans available to a child below the morally permissible threshold. Let us consider cloning first. In so far as the life plans available to children depend on their physical and mental abilities rather than on factors extrinsic to the child, eg, the wealth of the parents or the structure of society and in so far as such physical and mental abilities depend on the children's genome rather than on their developmental environment , the array of life plans available to a cloned child is similar to the array of life plans available to the person from whom the child's genome is derived.
Thus, unless the parents select for their child the genome of someone who, because of his or her genes, did not have a decent minimum number of different life plans from which to choose, the parental decision to conceive a child by cloning cannot in general interfere with the child's chances to have a reasonable array of life plans at his or her disposal.
In fact, if the parents decide to create a child by cloning someone who had many options and opportunities in life, their choice will in general positively contribute to the range of life plans available to the child. What about genetic engineering? Such genetic choices would in general enlarge rather than reduce the array of life plans available to the future child. But some parents may want to use genetic engineering to increase the chances of their child becoming fit for a very specific life plan.
In many cases, such genetic choices would reduce the range of life plans available to a child to the same extent as currently accepted environmental choices such as the decision to make a child play a lot of tennis. In other cases, the genetic choices would reduce the range to the same extent as environmental choices that are currently considered illegitimate such as the decision not to send a child to school at all.
In so far as we can make sure that cases of the second kind are relatively rare, the possible occurrence of these cases cannot be used as a reason to ban the reproductive use of genetic engineering. Parents can in principle use cloning and genetic engineering to make their children unable to choose their life plan. They could choose for their children genes that interfere with the normal development of the cognitive and emotional abilities required to compare and select life plans or they could choose for their children genes that are likely to make them fit only for a very specific life plan.
It is for these reasons that, as in the case of parental environmental choices, parental genetic choices should be regulated and constrained. But they should be regulated and constrained in ways that are similar to the way in which environmental choices are regulated. Some commentators think that the array of life plans available to people depends not only on their skills and sociophysical context but also on the way with conceive of themselves and of the options available to them.
Such expectations would exert a powerful psychological pressure on the cloned person and would heavily restrict the array of life plans effectively available to him or her. In their view, genetically engineered people who know that their parents chose for them genes designed to bring about, say, a preference for a particular kind of career would feel heavily constrained in the kind of choices they can make, and parental expectations would amplify this feeling.
My reply in this case is similar to my reply to Habermas. We could also start teaching people from an early age that the fact that two people have the same genes does not imply that they are destined to live similar lives. Similar considerations apply, mutatis mutandis, to the case of genetically engineered people.
What about parental expectations? No good evidence supports this claim either. Parental expectations often have an important and positive role in child development, even though they can occasionally harm a child and unjustly constrain his or her future freedom of choice. It should also be noticed that we currently accept very strong and pressing parental expectations as legitimate.
Consider the example of the firstborn royal child, who has to cope not only with the expectations of the parents but also with the expectations of a whole nation and beyond: everybody expects the child to take the throne when the right moment comes. This suggests that we do not usually see such expectations as infringing on the royal child's right to an open future.
Some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies undermine the autonomy of the resulting child. In this paper, I have considered two versions of this objection. I have argued that there is no evidence that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions.
And I have argued that there is no evidence that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans. But there seem to be no asymmetry between parental genetic choices and parental environmental choices with respect to the ways, the circumstances and, arguably, the frequency with which these choices would be used to perpetrate abuse.
Thus, no such asymmetry can be appealed to in arguing that the restrictions on parental genetic choices and on the biotechnologies that make such choices possible should be much more severe than current restrictions on parental environmental choices. From a legislative viewpoint, genetic choices and environmental choices should be treated in similar ways. Many issues remain unresolved. One issue concerns the best ways to avoid parents from using cloning and genetic engineering to perpetrate abuse.
We also need to establish how to distinguish in a principled way between genetic interventions that constitute abuse and genetic interventions that do not. These issues are important, but they should be seen as special cases of general questions about the permissibility of producing certain kinds of effects on children, whether through genetic interventions or through environmental interventions. The fact that a given intervention affects a child's genome rather than his or her environment does not make that intervention more likely to constitute abuse.
And the fact that an abuse has been perpetrated by a choice of genes does not make the abuse worse or its effects more irreversible than if it had been perpetrated through an intervention on the child's developmental environment.
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