Xenotransplantation (xeno- from the Greek meaning "foreign") is the transplantation of living cells, tissues or organs from one species to another such as from pigs to humans (see Medical grafting). Such cells, tissues or organs are called xenografts or xenotransplants. The term allotransplantation refers to a same-species transplant. Human xenotransplantation offers a potential treatment for end-stage organ failure, a significant health problem in parts of the industrialized world. It also raises many novel medical, legal and ethical issues. A continuing concern is that pigs have different lifespans than humans and their tissues age at a different rate. Disease transmission (xenozoonosis) and permanent alteration to the genetic code of animals are also a cause for concern.
Because there is a worldwide shortage of organs for clinical implantation, about 60% of patients awaiting replacement organs die on the waiting list. Recent advances in understanding the mechanisms of transplant organ rejection have brought science to a stage where it is reasonable to consider that organs from other species, probably pigs, may soon be engineered to minimize the risk of serious rejection and used as an alternative to human tissues, possibly ending organ shortages.
Other procedures, some of which are being carefully investigated in early clinical trials, aim to use cells or tissues from other species to treat life-threatening and debilitating illnesses such as cancer, diabetes, liver failure and Parkinson's disease. If vitrification can be perfected it could allow for long-term storage of xenogenic cells, tissues and organs so they would be more readily available for transplant.
There are only a few published successful xenotransplant procedures.
Xenotransplants are transplants of organs from one species to another, such as an animal organ to humans. They are cutting edge of medical science and could save thousands of people’s lives who are waiting for an organ donation. The animal organ, probably from a pig or baboon could be genetically altered with human genes to trick a patient’s immune system into accepting it as apart of its own body. They have re-emerged because of the lack of organs available and the constant battle to keep immune systems from rejecting the organs. Xenotransplants are thus hopefully able to provide a way of transplants which are safe and effective.
Acceptance
Xenografts have been a controversial procedure since they were first attempted. Many, including animal rights groups, strongly oppose killing animals in order to harvest their organs for human use. Medical concerns exist about possible disease transfer between animals and humans, such as the porcine endogenous retrovirus found in pig tissues. Religious beliefs, such as the Jewish and Muslim prohibition against eating pork, have been sometimes thought to be a problem, however according to a Council of Europe documentation both religions agree that this rule is overridden by the preservation of human life.
In general, however, the use of pig and cow tissue in humans has been met with little resistance, save some religious beliefs.
Some of the main and biggest biological/human health problems involved with Xenotransplants are that of transmitting animal diseases to humans, the unknown certainty of an outbreak of infectious diseases and the probability of rejection of donor organs. Baboons and pigs carry myriad transmittable agents which are harmless in their natural host but extremely toxic and deadly in humans. HIV is an example of a disease which was believed to be transmitted to humans by monkeys. Scientists and researchers also do not know if an outbreak of infectious diseases could occur and if they could contain the outbreak even though they have measures for control. Another obstacle for Xenotransplants is that of the body’s rejection of foreign objects by it immune system. These antigens (foreign objects) are often treated with powerful drugs which may in turn make the patient vulnerable to other infections and actually aid the disease trying to be cured. This is the reason the organs would have to be altered to fit with the patients DNA. In 2005, the Australian National Health and Medical Research Council declared a eighteen-year moratorium on all animal-to-human transplantation, concluding that the risks of transmission of animal viruses to patients and the wider community have not yet been resolved . The main ethical issues associated with Xenotransplants are that the animals which would be commonly used for their organs, such as pigs and baboons are killed or sacrificed. Baboons are very similar to humans with human-like hands, faces and a developed social structure. For this reason pigs could be used more as their anatomies are similar to humans and are a lot easier to breed than baboons that only produce one offspring at a time. Pigs are also a lot healthier and carry less disease than primates as well. There are less moral objections to the killing of pigs as they are already killed for food and are already being produced.
Friday, September 18, 2009
Transcriptome
The transcriptome is the set of all messenger RNA (mRNA) molecules, or "transcripts," produced in one or a population of cells. The term can be applied to the total set of transcripts in a given organism, or to the specific subset of transcripts present in a particular cell type. Unlike the genome, which is roughly fixed for a given cell line (excluding mutations), the transcriptome can vary with external environmental conditions. Because it includes all mRNA transcripts in the cell, the transcriptome reflects the genes that are being actively expressed at any given time, with the exception of mRNA degradation phenomena such as transcriptional attenuation. The study of transcriptomics, also referred to as Expression Profiling, examines the expression level of mRNAs in a given cell population, often using high-throughput techniques based on DNA microarray technology. The use of next-generation sequencing technology to study the transcriptome at the nucleotide level is known as RNA-Seq .
Transcriptomics is the branch of chemistry that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type.
Applications and analysis
The transcriptomes of stem cells and cancer cells are of particular interest to researchers who seek to understand the processes of cellular differentiation and carcinogenesis. A number of organism-specific transcriptome databases have been constructed and annotated to aid in the identification of genes that are differentially expressed in distinct cell populations or subtypes; however, the analysis of relative mRNA expression levels can be complicated by the fact that relatively small changes in mRNA expression can produce large changes in the total amount of the corresponding protein present in the cell. One analysis method, known as Gene Set Enrichment Analysis, identifies coregulated gene networks rather than individual genes that are up- or down-regulated in different cell populations.
mRNA regulation
Although microarray studies can reveal the relative amounts of different mRNAs in the cell, levels of mRNA are not directly proportional to the expression level of the proteins they code for. The number of protein molecules synthesized using a given mRNA molecule as a template is highly dependent on translation-initiation features of the mRNA sequence; in particular, the ability of the translation initiation sequence is a key determinant in the recruiting of ribosomes for protein translation. The complete protein complement of a cell or organism is known as the proteome.
A study of 158,807 mouse transcripts revealed that 4520 of these transcripts form antisense partners that are base pair complementary to the exons of genes. These results raise the possibility that significant numbers of "antisense RNA-coding genes" might participate in the regulation of the levels of expression of protein-coding mRNAs.
Transcriptomics is the branch of chemistry that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type.
Applications and analysis
The transcriptomes of stem cells and cancer cells are of particular interest to researchers who seek to understand the processes of cellular differentiation and carcinogenesis. A number of organism-specific transcriptome databases have been constructed and annotated to aid in the identification of genes that are differentially expressed in distinct cell populations or subtypes; however, the analysis of relative mRNA expression levels can be complicated by the fact that relatively small changes in mRNA expression can produce large changes in the total amount of the corresponding protein present in the cell. One analysis method, known as Gene Set Enrichment Analysis, identifies coregulated gene networks rather than individual genes that are up- or down-regulated in different cell populations.
mRNA regulation
Although microarray studies can reveal the relative amounts of different mRNAs in the cell, levels of mRNA are not directly proportional to the expression level of the proteins they code for. The number of protein molecules synthesized using a given mRNA molecule as a template is highly dependent on translation-initiation features of the mRNA sequence; in particular, the ability of the translation initiation sequence is a key determinant in the recruiting of ribosomes for protein translation. The complete protein complement of a cell or organism is known as the proteome.
A study of 158,807 mouse transcripts revealed that 4520 of these transcripts form antisense partners that are base pair complementary to the exons of genes. These results raise the possibility that significant numbers of "antisense RNA-coding genes" might participate in the regulation of the levels of expression of protein-coding mRNAs.
Human Insulin
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. 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. 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" 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." The two protein chains were then combined to produce insulin molecules.
Human Growth Hormone
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, creating significant shortages of the hormone. 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" 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." Then "a chemically synthesized DNA 'adaptor' fragment containing an ATG initiation codon..." 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." 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" which might be difficult to remove.
Human Blood Clotting Factors
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...
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. 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.
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:
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 and the entire 3'-untranslated sequence.
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.
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" 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...
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.
Human Growth Hormone
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, creating significant shortages of the hormone. 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" 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." Then "a chemically synthesized DNA 'adaptor' fragment containing an ATG initiation codon..." 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." 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" which might be difficult to remove.
Human Blood Clotting Factors
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...
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. 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.
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:
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 and the entire 3'-untranslated sequence.
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.
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" 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...
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.
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