I need to write a 1 page essay for the following article and need major help. Th
ID: 183683 • Letter: I
Question
I need to write a 1 page essay for the following article and need major help. The essay must explain how animals are genetically altered for use in research and how these animals have been used to help study and hopefully alleviate human disease.
2.3 How are ACHM used in research?
2.3.1 Genetically altered animals in investigating health and disease
The DNA sequence of many species is sufficiently similar for sections from one species to retain their function when incorporated
into cells of a different species. In a classic experiment, human DNA was inserted into mutant yeast cells defective in a gene (cdc2) known to be crucially important in regulating yeast cell division. Remarkably, some pieces of human DNA were able to compensate for the defective yeast gene, allowing the mutant cells to divide normally. Researchers thus identified the human cdc2 gene, which is so similar that it could compensate for the defective yeast gene.31 These experiments were important in demonstrating that some genes responsible for controlling basic cell functions like cell division are highly conserved (meaning they have retained the same structure and function throughout evolution). The process of cell division is fundamental to understanding cancer, and variants of the cdc2 gene are associated with some forms of human cancer. (See Box 2.4 for uses of genetically
altered cells.)
It is now almost routine to incorporate human DNA into animal eggs or embryos; the
resulting genetically altered animals are used ubiquitously in research to investigate the function of human genes and the proteins they encode. For example, the melanocortin receptor (MC1R) regulates pigmentation in mammals and is necessary for the production of dark melanin pigment in skin and hair. Humans
with certain MC1R variants have red hair, pale ultraviolet-sensitive skin and are at increased risk of skin cancer. Mice expressing these human MC1R variants have yellow coats, and have been used to study the activation of MC1R receptors, and to identify the cell signalling pathways through which they work.32
Where the genetic basis of a disease in humans is known or suspected, the particular variant of the human gene associated with the disease can be incorporated into an animal to study the disease (see Box 2.3). We received many submissions describing the use of mice expressing human genes to study conditions as varied as migraine, anxiety disorders, osteoporosis, diabetes, heart disease and cancer.33 However, the use of a wider range
of species was also evident, including fruit flies expressing human ion channels used to study neurodegenerative disorders, and pigs expressing human polypeptide receptors in diabetes research.34,35
31 Lee MG, et al. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327 (6117), 31–5.
32 Jackson IJ, et al. (2007). Humanized MC1R transgenic mice reveal human specific receptor function. Hum Mol Genet 16, 2341–8.
33 Eikermann-Haerter K, et al. (2009). Androgenic suppression of spreading depression in familial hemiplegic migraine type 1 mutant mice.
Ann Neurol 66, 564–8; Jennings KA, et al. (2006). Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission. J Neurosci 26, 8955–64; Daley E, et al. (2010). Variable bone fragility associated with an Amish COL1A2 variant and a knock-in mouse model. J Bone Miner Res 25, 247–61; King M, et al. (2008). Humanized mice for the study of type 1 diabetes and beta cell function. Ann N Y Acad Sci 1150, 46–53; Su Q, et al. (2008). A DNA transposon-based approach to validate oncogenic mutations in the mouse. Proc Natl Acad Sci USA 105, 19904–9.
34 Moffat KG (2008). Drosophila genetics for the analysis of neurobiological disease. SEB Exp Biol Ser 60, 9–24.
22 35 Renner S, et al. (2010). Glucose intolerance and reduced proliferation of pancreatic ß-cells in transgenic pigs with impaired glucose-dependent
insulinotropic polypeptide function. Diabetes 59, 1228–38.
2 RESEARCH INVOLVING INTER-SPECIES MIxTURES
Box 2.3 Examples of research methods used to make genetically altered animals
1. Transgenesis can be achieved in a wide range of species, using methods including:
DNA microinjection. Copies of a segment of (e.g. human) DNA are directly injected
into the nucleus of a fertilised animal egg, which is gestated in a surrogate female.36 The genomes of the offspring are analysed, and animals in which the injected DNA has integrated are bred for use. DNA insertion occurs at random, and often in multiple copies. Genes within the introduced DNA can be expressed in a manner that is expected, or they can show ectopic (out of place) expression depending on the site of integration. In a minority of cases the integration event can disrupt the activity of an endogenous gene.37
Retrovirus-mediated gene transfer. A modified carrier virus (or ‘vector’) is used to insert a transgene into the cells of a developing embryo, which is gestated in a surrogate female. The resulting offspring are often genetic ‘mosaics’, developed from a mixture of cells with one or more copies of the inserted sequence at different places in their genomes. Animals where the germ cells have the required integrated DNA are bred to create transgenic animal strains. Recent studies indicate that it may be possible to generate transgenic NHPs in this way.38
2. Gene-targeting methods include:
• Homologous recombination in embryonic stem (ES) cells is used to engineer
precise changes in the mouse genome.39 ES cells are genetically modified in vitro, e.g. to add, remove or exchange a specific genetic sequence at a specific location in the genome. Individual cells can be selected that following rare DNA recombination events, have the intended changes to their DNA.40,41 These cells are injected into early stage mouse embryos to make chimæras. Mice with germ cells developed from the altered ES cells are bred, to create a line of genetically altered mice.
These methods in the mouse have become very sophisticated. Similar techniques are being developed in other species (see 3.2). In theory it ought to be possible make chimæras with NHP ES cells (which have very similar properties to human ES cells, distinct from those of the mouse) and NHP embryos, though this has not yet been attempted to our knowledge.42 It is not clear whether human pluripotent cells can contribute to pre-implantation human embryos to make chimæras.43 (Additional methods of transgenesis and gene targeting see 44)
3. Somatic cell ‘gene therapy’.Techniques have been developed to integrate transgenes into particular somatic tissues (such as immune cells, the lung or retina). These methods often use modified viruses as ‘vectors’ to carry sections of DNA into the cells of adult animals or humans, rather than embryos. These methods generally involve gene addition rather than replacement, with the purpose of restoring the function of an abnormal gene.
36 Gestation in a surrogate is used for research involving mammals; the embryos of other genetically altered species, including chick, frog and fish can develop by themselves.
37 For an overview see Gama Sosa MA, et al. (2010). Animal transgenesis: an overview. Brain Struct Funct 214, 91–109.
38 Niu Y, et al. (2010). Transgenic rhesus monkeys produced by gene transfer into early-cleavage-stage embryos using a simian
immunodeficiency virus-based vector. Proc Natl Acad Sci USA 107, 17663–7.
39 The types of change can include deletions, insertions, or replacement of one DNA sequence with another. These methods rely on the use of
DNA sequences, at the ends of the donor DNA that are homologous to (match) the target site in the ES cell genome.
40 While DNA usually integrates at random in mammalian cells, even rare homologous recombination events can be found by screening large
numbers of ES cells.
41 Gordon JW, et al. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77, 7380–4.
42 Wianny F, et al. (2011). Embryonic stem cells in non-human primates: An overview of neural differentiation potential. Differentiation
81, 142–52.
43 Although the HFE Act (2008) would allow these experiments to be initiated, it would be illegal to keep such entities intact in vitro for more
than 14 days or to implant them (see Box 6.5).
44 a. Sperm-mediated gene transfer. Can also be used to create transgenics. A sequence of DNA is introduced into the head of a sperm, which
is then used for fertilisation. This approach has been used in species including frog, mouse, rat and pig.
b. Genetic alteration of somatic cells combined with nuclear transfer. In species for which ES cells are unavailable (e.g. sheep) gene
targeting can be conducted by combining the use of somatic cells (e.g. fibroblasts) genetically modified in culture, with nuclear transfer cloning
techniques. See Denning C, et al. (2001). Gene targeting in primary fetal fibroblasts from sheep and pig. Cloning Stem Cells 3, 221–31.
c. Zinc-finger nuclease (ZFN) methods. These methods can be used on cells in culture, or after DNA microinjection into fertilised eggs.
In principle this method can be used to introduce human DNA into any animal species and in a targeted fashion. See Whyte JJ, et al. (2011).
Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs. Mol Reprod Dev 78, 2.
d. Genetic modification of spermatogonial stem cells. Male germ-line (spermatogonial) stem cells can be genetically modified and
transplanted into the testicular tissue of an infertile male animal where they give rise to modified sperm cells. This approach has been
developed in the mouse. See Takehashi M, et al. (2010). Generation of genetically modified animals using spermatogonial stem cells. 23 Dev Growth Differ 52, 303–10.
ANIMALS CONTAINING HUMAN MATERIAL
Box 2.4 Transgenic and genetically altered cells
Individual animal cells, or cell lines, into which human genes are inserted (or ‘transfected’) are widely used in investigational research and drug development.
Expression of human DNA in frog eggs has been used to understand the function of some human transporter proteins (molecules that move substances into and out of cells). One of the first demonstrations of the chloride channel function of the cystic fibrosis gene was achieved using this approach.45 More recently, suggestions arose of an association between variants
of the human gene SLC2A9 with high uric acid levels in gout. Human SLC2A9 was initially thought to encode a protein used only to transport sugars; however, its expression in frog eggs revealed a new role for the transporter in carrying uric acid, and suggested a rationale for the links between human SLC2A9 gene variants and gout.46
Transfected cells lines expressing human genes are also used in the pharmaceutical industry in screening to identify novel drug molecules, and to express human proteins (marketed products include human erthyropoetin for use in anaemia, and blood clotting factors for use in haemophilia, produced in Chinese hamster ovary cells).47
(See also 2.2.3 for the uses of inter-specific cell hybrids.)
Huntington’s disease (HD) is a genetic neurodegenerative condition, in which nerve cells in some parts of the brain accumulate granular protein and subsequently die. Animal models of HD have been created in flies, zebrafish, mice and sheep by incorporating the mutant form of the human Huntingtin gene, which causes HD in man, into the animals’ genomes.48,49 A rhesus macaque transgenic model of the disease was also reported in 2008, although the mutant human Huntingtin gene did not transmit to offspring.50
Studies using cell cultures and these animal models indicated that the abnormal granular protein product of the mutant Huntingtin gene, which is toxic to brain cells, could be cleared by a process called autophagy. Drugs that induce autophagy were identified, and found to enhance the removal of the protein and thus decrease its toxicity. The consistent effect of this strategy in the animal models of HD
suggested that a drug might similarly modify the accumulation of the toxic protein granules in human brain cells. Safety testing of one these drugs is now underway, as a precursor
to clinical trials in patients.51 Autophagy has also been implicated in other diseases including Parkinson’s, Alzheimer’s, and forms of cancer
– some of the evidence for this association comes from comparable studies in transgenic mice expressing the human proteins mutated in these diseases.
The study of Duchenne muscular dystrophy (DMD), a condition that causes progressive muscle wasting in boys leading to death in early adulthood, has been facilitated by genetically altered animals expressing human gene variants. A mouse was first discovered that carried a dystrophin gene mutation similar to that causing DMD in humans.52 Although the mouse had some biochemical and physical features of DMD, it lacked the characteristic
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45 46
47
48 49 50 51
Bear CE, et al. (1991). Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J Biol Chem 266, 19142-5.
Vitart V, et al. (2008). SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout.
Nat Genet 40, 437-42.
See the European Medicines Agency http://www.ema.europa.eu/; EMEA/H/C/000726 epoetin alfa for the treatment of anaemia; EU/3/09/655: Human recombinant octocog alfa for the treatment of haemophilia A.
Williams A, et al. (2008). Novel targets for Huntington‘s disease in an mTOR-independent autophagy pathway. Nat Chem Biol 4, 295–305. Jacobsen JC, et al. (2010). An ovine transgenic Huntington‘s disease model. Hum Mol Genet 19, 1873–82.
Yang SH, et al. (2008). Towards a transgenic model of Huntington‘s disease in a non-human primate. Nature 453, 921–4.
Rose C, et al. (2010). Rilmenidine attenuates toxicity of polyglutamine expansions in a mouse model of Huntington‘s disease. Hum Mol Genet 19, 2144–53.
52 Bulfield G, et al. (1984). X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 81, 1189–92.
severe early onset, and so did not fully mimic human DMD. A second gene, utrophin, was later identified and found to have a very similar function to the dystrophin gene. Although the utrophin gene is inactivated early in embryonic life in humans, mice can partially re-activate this gene in adulthood, compensate for an absence of dystrophin, and ameliorate the effects of DMD. Mice genetically altered to
lack the function of both genes show severe disease and more closely model human DMD. Research using mouse models has since led to the development of several putative DMD treatments, including an approach which partially corrects the genetic defect in many cases of DMD, now in clinical trial.53
A strain of dog with a spontaneous dystrophin mutation has also been used in DMD research.54 Large animal models are not always needed in disease research, and pre- clinical research in such species including dogs is not necessarily a pre-requisite for drug development. However, conditions such as heart disease and cognitive dysfunction may require large animal models because of the significant biological differences between man and mouse; humanised animal models may in future be of use in the development of therapies for such diseases.
While many human diseases (e.g. HD, DMD) are caused by mutations in protein coding regions of DNA, disease-causing mutations also occur in DNA regulatory regions (which do not encode protein but regulate gene expression). Regulatory regions are often located at a considerable distance from the genes they control, and the creation of accurate animal disease models involving mutations in these regions therefore requires the transfer of extensive sections of DNA (see the modification
of -globin gene locus used to model the blood disorder -thalassaemia in the mouse in 3.2). The study of human gene regulatory regions in transgenic animals (mice, chick, embryos, frogs and fish), combined with detailed sequence comparisons, has also led to basic understanding of how these function normally, or are defective in genetic disease, and how they and the gene regulatory mechanisms have evolved.55,56,57 We anticipate that it will become increasingly possible to accurately manipulate large sections of human DNA.
2.3.2 Genetically altered animals used in developing and testing therapeutics Animals containing human genetic sequence can be developed to produce humanised substances (e.g. proteins and antibodies) for use as ‘biological therapeutics’ in people with deficiency of a particular substance, or in other forms of novel treatment.58
In an approach sometimes called ‘pharming’, transgenic animals have been created which carry a human gene, and secrete the associated human protein e.g. as a component of their milk. The protein is extracted, purified and used for treatment. Such ‘therapeutic proteins’ have been produced in sheep, goats, cattle, and rabbits; chickens have been developed which produce human proteins in their egg white.59
In 2009, ATryn, a human anti-thrombin protein made by transgenic goats was licensed for use during surgery in patients with a congenital blood clotting disorder.60 Similar products in development include human -1 antitrypsin
for emphysema treatment, and blood clotting factors for haemophilia treatment. In these approaches the genetically altered animals are, in effect, used to manufacture often large amounts of fully functional proteins, which cannot be produced in cell lines.
2 RESEARCH INVOLVING INTER-SPECIES MIxTURES
53 Kinali M, et al. (2009). Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol 8, 918–28.
54 To date these have not been used extensively in therapeutic drug development.
55 For an example of an early transgenic experiment see Koopman P, et al. (1989). Widespread expression of human alpha 1-antitrypsin in
transgenic mice revealed by in situ hybridization. Genes Dev 3, 16–25.
56 For an example of a recent paper involving a systematic study of regulatory sequences see Schmidt D, et al. (2010). Five-vertebrate ChIP-seq
reveals the evolutionary dynamics of transcription factor binding. Science 328, 1036–40.
57 For an example of a recent work considering loss of regulatory sequences in human evolution see McLean CY, et al. (2011). Human-specific
loss of regulatory DNA and the evolution of human-specific traits. Nature 471, 216–9.
58 Biological therapies are treatments for diseases that involve the use of biological materials or biological response modifiers, such as genes,
cells, tissues, organs, serum, vaccines, antibodies or humoral agents. In contrast, pharmacological or chemical therapies are those which use
small drug molecules.
59 Written evidence from the Biotechnology and Biological Sciences Research Council (BBSRC), and see for example Lillico SG, et al. (2007).
Oviduct-specific expression of two therapeutic proteins in transgenic hens. Proc Natl Acad Sci USA 104, 1771–6.
60 The European Public Assessment Report (EPAR), produced by the European Medicines Agency for ATryn is available at 25
http://www.ema.europa.eu/humandocs/Humans/EPAR/atryn/atryn.htm
ANIMALS CONTAINING HUMAN MATERIAL
Humanised antibodies produced in animals are increasingly used as biological therapeutics. Animals produce a huge range of different antibodies which underpin the immune recognition and rejection of ‘foreign’ proteins (‘the adaptive immune response’). Each antibody interacts highly specifically with a particular protein. This ability has been used
to develop ‘therapeutic antibodies’ in which an antibody can act directly as a ‘biological drug’ by blocking some cellular function or killing
the cell type targeted (e.g. cancer cells); or can be coupled to a drug which the antibody delivers to a specific target. This field is fast- growing; in mid-2009, there were close to 50 approved therapeutic antibodies on the market, and over 150 applications for new antibody products under consideration in the USA.61 Antibodies are large, complex proteins, which are difficult to produce synthetically, but they can be obtained from animals or certain cell lines. However, animal antibodies injected
into humans would be recognised as ‘foreign protein’ and eliminated by the human immune system. Recently, mice with ‘humanised immune systems’ have been engineered to produce antibodies that are not rejected by the human body, and so can be used in therapy.62 This has been achieved using mice with antibody genes replaced by human equivalents (e.g. xenoMouse, see also 3.2).63 In response to immunisation the mouse humanised immune systems respond by producing humanised antibodies, which can be selected and manufactured in cell lines. The human antibody Panitumumab, licensed for colorectal cancer treatment, was developed in this way. It targets a growth factor receptor, and inhibits tumour growth and vascularisation.64
The concept of ‘gene replacement therapy’ was first discussed in the early 1970s, but safe, effective procedures have proved difficult to develop. Gene therapy is based on the concept of inserting a functional copy of a gene into tissues where the gene is dysfunctional or absent (see Box 2.2). The aim is to perform human–human gene transfer; however, animal models are necessary to develop and refine the required reagents and techniques.
Leber congenital amaurosis (LCA) is a set
of genetic eye diseases which often lead to complete blindness. One form of LCA is caused by a mutation in the RPE65 gene, which encodes a protein needed for the recycling
of visual pigment in the eye’s light-sensing cells. Gene therapy aims to carry functional copies of the RPE65 gene into the retina using
a modified viral carrier introduced into the eye.65 These methods have been developed in transgenic mice with a defective RPE65 gene and in the Briard dog which naturally lacks the RPE65 gene.66 Both the mouse and dog models have early, severe visual impairment similar
to that in human LCA; however, the dog eye
is more similar to the human eye in size and structure. The effectiveness of this therapy in these animals enabled the approach to be taken forward into clinical trials; initial results suggest that it can be effective in humans, though further refinement will be required to produce
a licensed treatment.67,68 This approach may
in future also turn out to be applicable to other eye diseases. There are particular sensitivities in using ‘companion’ animals such as dogs and cats for experimental purposes, but there are some unusual situations where they have clear advantages (either because of some aspect of
61 Nelson AL, et al. (2010). Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 9, 767–74.
62 Kyowa Hakko Kirin California, Inc. have developed the TransChromo Mouse (TC MouseTM) that is capable of producing a variety of fully human
monoclonal antibodies. They are also developing the TransChromo Cow (TC CowTM) for the production of polyclonal antibodies.
See: http://kyowa-kirin-ca.com/tc_pubs.cfm
63 Jakobovits A, et al. (2007). From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice.
Nat Biotechnol 25, 1134–43; Written evidence from the NC3Rs.
64 Giusti RM, et al. (2007). FDA drug approval summary: panitumumab (Vectibix). Oncologist 12, 577–83.
65 Acland GM, et al. (2001). Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 28, 92–5.
See also http://www.ucl.ac.uk/ioo/research/patients/clinical_trials.html
66 Bemelmans AP, et al. (2006). Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital
amaurosis. PLoS Med 3, e347.
26 67 Bainbridge JW, et al. (2008). Effect of gene therapy on visual function in Leber‘s congenital amaurosis. N Engl J Med 358, 2231–9.
68 Maguire AM, et al. (2008). Safety and efficacy of gene transfer for Leber‘s congenital amaurosis. N Engl J Med 358, 2240–8.
their normal function or, as here, because of the presence of a naturally occurring disease which closely resembles a human disorder) as to outweigh this aversion. Animal models are also contributing to attempts to develop gene therapies for conditions including spinal muscular atrophy and -thalassemia.69,70
Owing to a shortage of human donor organs, tissue from animals, particularly pigs, has for many years been investigated as a source of tissue for transplant, although safety concerns hampered the development of the field. Another major barrier to the xenotransplantation of organs from pigs to humans is the ‘hyperacute immune response’ in which the recipient’s immune system destroys the lining of blood vessels in the engrafted tissue. Such rejection occurs in part because an antigen (alpha-Gal), which is not made by humans, is expressed
on the surface of pig cells. Attempts are under way to develop pigs which do not express alpha-Gal.71 An alternative approach is the development of transgenic pigs expressing critical human proteins which inhibit the human immune response, and whose organs are therefore less likely to be rejected. Evidence from pre-clinical studies has indicated the potential of this approach, for example hearts from transgenic pigs have been found to function following transplant into NHPs treated with immunosuppressive drugs.72
Transgenic mice may, in future, be used in drug-toxicity testing and in testing biological products such as live vaccines. These are avenues in which the use of humanised animals may reduce, or ultimately replace,
the use of larger animal species. However, the development of such methods can take several decades, not only for the necessary scientific development, but in subsequently gaining acceptance from regulatory agencies.73
Explanation / Answer
In order to use animals for research, it is very important that they should be altered from the pair genes so that the effect that one desires could be determined. In many cases, some genes of the human DNA have been identified, which are incorporated in the animals so that the functions of these genes could be identified in a better manner. This results in altered functions in these genetically altered species and allows the researchers to study the effects of these genetic alterations in the species. This is done when a disease is suspected that might be having a genetic basis. Once this is done, the full spectrum of the action of the gene causing the disease could be identified.
There are a number of methods for alteration of the animals on a genetic basis that includes micro injection Where, human DNA segment would be directly injected in the nucleus of a fertilised animal egg, that would be gestated in a surrogate female. When genes would be introduced in the DNA as these are expressed better. Retrovirus mediated transfer is where the virus is inserted the transgene of the cell. Transgenic animal strains are created when the germ cells have the required DNA. Somatic gene therapy is used to integrate genes into somatic cells where modified vectors are integrated into somatic cells so that the normal function of the cell can be restored. For species including frog, mouse, rat and pig, spe-rm mediated transfer is used where the DNA is introduced in the head of the sperm.
For drug research, animals are frequently used, where one of the first demonstrations was done for cystic fibrosis. For expressing human proteins, transfected cell lines are frequently used. For Huntington disease, animal models have been used a s drugs inducing autophagy were identified.