What Are Some Of The Issues That Genetically Modified Animals Come Across?

Abstract

Genetically engineered animals take opened new frontiers in the report of physiology and affliction processes. Mutant animals offer more accurate illness models and increased precision for pathogenesis and handling studies. Their utilise offers hope for improved therapy to patients with atmospheric condition that currently have poor or ineffective treatments. These advantages take fostered an increase in studies using mice in recent years, a development viewed with alarm by those who oppose the employ of animals in research. Scientists bespeak out that the mice are replacing more sentient species, such as nonhuman primates, and are increasing the quality of enquiry existence conducted. They assert that study of genetically engineered animals will somewhen permit decreases in numbers of animals used in inquiry. Nevertheless, the increase in use of genetically altered animals presents many challenges in reviewing protocols and providing intendance. Identification and resolution of any welfare problems is a responsibleness that is shared by institutional animate being care and employ commission, veterinary, animal care, and research staffs. To place potential welfare concerns, a database such as TBASE (<http://tbase.jax.org>) can be searched to learn what has been reported for established mutant lines. In addition, newly created lines should be monitored by a surveillance arrangement and accept phenotype cess to identify the effects of altering the genome. Methods of ensuring welfare can include treatment of conditions produced, restriction of gene expression to tissues of interest or to sure fourth dimension periods, and establishment of endpoints for removing animals from a study before problems appear.

Introduction

Modification of the genome of animals has occurred throughout the ages. Initially, changes in genetic limerick occurred spontaneously. With the beginning of agronomics, humans exerted influence on the procedure by selecting animals with desirable genetic traits. An beast with a spontaneous genetic mutation that increased feed conversion, boosted milk production, or produced more desirable carcass characteristics would exist selected as breeding stock to improve the herd by perpetuating advantageous phenotype changes. More recently, scientists accelerated the mutation procedure with irradiation and chemic mutagens. Many genetic loci were identified, mapped, and studied using such tools. Genetic predisposition and resistance to diseases were detected.

The era of transgenic animals is a relatively new development that resulted from the injection of foreign Dna into the pronucleus of embryos ( Gordon et al. 1980 ; Palmiter et al. 1982 ). Offspring expressed the injected Dna construct and passed it on to succeeding generations. The ability to dispense and report the genome has increased profoundly with the subsequent development of factor targeting technology, which allows an investigator to knock out a cistron sequence of involvement in embryonic stem (ES i ) cells ( Capecchi 1989 ). Point mutagenesis accomplished by administration of N-ethyl-N-nitrosourea (ENU 1 ) is some other tool in widespread employ to create new disease models.

This commodity discusses some of the areas of research that are aided past the cosmos of these animals, welfare issues that have been associated with their production, sources of information to help predict problems, tools available to prevent or limit any welfare difficulties that occur, issues in institutional animate being care and use commission (IACUC 1 ) review of studies of genetically altered animals, and strategies for addressing welfare bug. Discussion of animal welfare bug generally includes the 3Rs (refinement, reduction, and replacement) of Russell and Burch (1959) . Proponents of the use of genetically engineered animals assert that they offer some unique advantages over normal or wild-type animals in achieving the 3Rs. They offering more authentic models that provide more than precise data than previously used paradigms, allow the replacement of higher species with less sentient species, and may ultimately reduce the number of animals required to address a detail affliction problem. These potential advantages are examined throughout this discussion.

Types of Inquiry

The mapping of the human genome is stimulating one of the nigh exciting periods in the history of biomedical research. Studies are in progress that could develop new ways to treat disease conditions past altering the genome. The prospect of such treatments has angry controversy regarding the ideals of potential treatments that would modify the man germ line and, thus, be passed to succeeding generations ( Editorial 1999 ; Willgoos 2001 ). Until the controversies are resolved, gene therapy trials in humans will probably exist limited to modalities that change genes only in somatic cells. These alterations would not affect offspring of treated patients.

In the near futurity, germline-modifying therapies volition probably be limited to laboratory animals. The ability to dispense the genome of animals makes them an indispensable component of enquiry involving alteration and modification of genes. It is expected that the research will produce unprecedented breakthroughs in medicine, simply a few of the genetically contradistinct animals created may present challenges to the IACUC, the veterinary staff, and others striving to ensure animal well being. Among the areas studied are gene discovery, affliction models, test systems, gene therapy, xenotransplantation, and life span extension.

Gene Discovery

Gene discovery is an expanse of investigation designed to determine the structure and office of various genes. A closely related expanse is proteomics, in which proteins produced by genes are studied to discover their activity and the interactions with other proteins in the body. Some of these studies are genome driven and employ lines of mice created with human or animal genes of interest existence either expressed or inactivated and determining the resultant phenotype. Other studies are phenotype driven and use mutagens such equally ENU and chlorambucil to produce animals with contradistinct phenotypes. These animals are then studied to determine the gene alterations that produced the particular phenotype.

Because many of the genes to be studied are necessary for fetal evolution or the basic functions of life, some of the animals produced will likely have wellness bug and increased lethality. Ensuring animal welfare in some of these newly created lines may require vigilant surveillance and innovative management.

Disease Models

Application of genetic technology techniques has enabled the creation of new models for human diseases that have previously lacked accurate spontaneous or experimentally induced animate being models. Conditions such equally Alzheimer'southward affliction, amyotrophic lateral sclerosis (ALS 1 ), and Parkinson's disease are subjects of intense efforts to develop new models. One instance is a line of transgenic mice, B6SJL-Tg N (SOD1-G93A) 1Gur, which overexpress mutant Cu, Zn superoxide dismutase and are proposed equally a model of ALS ( Gurney et al. 1994 ). The mice develop a lower motor neuron syndrome characterized by hind limb weakness at iii to 4 mo of age that progresses to paralysis by v mo. The syndrome is similar to that seen in human patients with ALS. Fifty-fifty inquiry for diseases that previously had many models, such equally diabetes mellitus, has been aided past new models adult using transgenic and knockout technology ( Wong et al. 1999 ). The development of improved models that offer advantages over existing ones volition continue and volition improve the quality of research. Because the lines created will manifest signs similar to those seen in humans afflicted by the same syndrome, they may present challenging welfare bug.

Piece of work is in progress to develop new models past altering the susceptibility of mice to pathogens of humans. Because the simply models for weather such as viral hepatitis and AIDS have been in nonhuman primates, the apply of genetically contradistinct mice offers the potential animal welfare improvement of performing the research in a less sentient species. There is some concern that genetically altered mice might provide a reservoir for these human pathogens and spread infection to the man population. Even so, the risk from the mice is not greater than from similarly affected nonhuman primates. It is crucial to prevent escape of altered animals to preclude breeding with wild animate being populations.

Inquiry into the genetic influence on tumor promotion and suppression is being performed in models created by overexpression or inactivation of specific genes. A group of models has been created by knocking out the transforming-related protein 53 (Trp53 1 or p53 ane ) tumor suppressor cistron ( Donehower et al. 1995 ; Harvey et al. 1993 ; Purdie et al 1994 ). These lines experience an increased incidence of tumors compared with their wild-type relatives. The frequency, historic period of onset, and even tumor type varies with different background strains. Commonly used strains for these models include C57BL/6 and 129/P.

Information technology is of import to assess the phenotype of each new line created to find new illness models. An example of the importance of examining each new line is illustrated past a particular strain, C57BL/6J-TgN(LckIL4)1315Dbl, of transgenic mice for interleukin-4 ( Lewis et al. 1993 ). In dissimilarity to lines previously created with similar constructs, one line (no. 1315) became progressively humpbacked starting at 3 to 6 mo of age. Phenotype cess revealed that the animals were afflicted with osteoporosis. This unique line became a valuable model for studying the previously unrecognized part of interleukin-iv in osteoporosis.

Exam Arrangement Development

Genetic engineering engineering science is being used to develop improved test systems for examining safety and toxicity of chemicals, products, drugs, and devices. New mouse models volition enable replacement of more than sentient species, such as nonhuman primates, in some of these assessments. For example, transgenic mice expressing the human polio virus receptor accept been created, and they testify promise as substitutes for nonhuman primates to test the safe of adulterate polio vaccines ( Ghendon and Lambert 1996 ). Carcinogenicity testing increasingly uses genetically modified animals, which offer improved systems for evaluation of compounds. Mice with activated myc, ras , and neu oncogenes provide test systems with increased sensitivity for the detection of carcinogenic chemicals.

As gene therapy modalities are developed, new prophylactic testing strategies volition need to be adult to assess the effects of the transgene existence delivered as well equally any toxic furnishings of vector systems used for delivery. The US Nutrient and Drug Assistants recognizes that animal models created through genetic or pharmacological means can be used in an effort to demonstrate that gene therapy products can correct a genetic defect, slow progression of a illness, or alleviate its signs ( Pilaro and Serabian 1999 ).

Gene Therapy

Treating disease conditions by altering the genome of somatic cells of affected humans and animals is an exciting area of research that provides hope for weather condition that shortly lack effective treatment modalities. Examples of changes existence investigated include replacement of a defective cistron with a normal one, insertion of resistance genes, or altering regulatory sequences to turn genes on or off. Enquiry in this surface area involves cosmos of laboratory animals with specific genetic defects then correcting the defects by therapy with normal genes. In reviewing these protocols, information technology is important to consider the welfare bug of untreated control groups.

Xenotransplantation

The demand for transplantable organs is fostering research into the creation of animals whose tissues are compatible for implanting into humans. Larger species, such every bit pigs and baboons, are preferred for development every bit donors because of the similarity of their organ size to that of humans. Notwithstanding, mice are being used in preliminary studies to assess feasibility and establish procedures to make the animal tissues uniform. These studies might produce animals with modified immune systems that are susceptible to a variety of organisms, some of which are presently believed to be nonpathogenic for the species. These animals may nowadays some novel diagnostic and therapeutic challenges.

Life Span Extension

As medical research has progressed, the advances in control of disease take resulted in a steady increase in longevity for humans, with increased health and vigor for the elderly. Contempo results with genetically contradistinct animals are raising hopes that perhaps life span tin exist increased even farther. One development that perpetuates such hope is a line of mice with a proto-oncogene at the SHC locus knocked out. (The gene designation SHC was identified by Pelicci and colleagues [1992] .) The p66 ^shc-/- mice have increased resistance to oxidative stress and a 30% increased life span compared with wild-blazon mice ( Migliaccio et al. 1999 ).

The demonstration of such genetic alterations that effect in increases in life span is a powerful incentive for enquiry into the genetic control of aspects of the aging process. This area of investigation will remain active in the nearly future. In this search for genes that will increase longevity, in that location volition as well be the creation of some lines with shorter life bridge and welfare bug.

Identification of Welfare Concerns

Welfare Issues in Production and Breeding

One of the most controversial and vexing issues associated with the employ of genetically engineered animals derives from the sharp increase in numbers of mice used in recent years. Opponents of their use assert that any welfare gains offered past refinement and replacement of other models are offset past the failure to reduce the total number of animals used. The increase in number of mice used in research in recent years can be attributed to intensified efforts in areas that were previously hampered by a lack of acceptable models, also as the evolution of new technologies.

Although accurate estimates for the number of mice used in research in the United States are non published, I believe that the expansion in mouse populations in centralized facilities at my institution over the past 8 year is consistent with what is occurring in similar institutions throughout the country. As illustrated in Figure one , the increase is a full of 161.3% or a rate of more than than 23% per year. The increase is due not only to growth in the numbers of animals on studies but also to the big number of animals necessary to create each genetically modified line. In transgenic and knockout lines, these animals include breeding males and donor females needed to produce the embryos for pronuclear injections and for harvesting blastocysts for injection of modified ES cells. Vasectomized males are needed to brood with females to produce pseudopregnant recipients for the altered embryos. In point mutagenesis studies, males are administered ENU to induce mutations in their spermatogonial stalk cells. Approximately one in 700 gametes will have a mutation at any given locus, with multiple contained mutations per offspring obtained. Depending on the screening protocol, <1% to up to 10% of animals volition take interesting phenotypes ( Balling 2001 ; Nelms and Goodnow 2001 ). Up to three generations of breeding and screening may be required to discover mutant progeny. A review of the methods required vividly illustrates the large number of animals used to establish and characterize the models created by this means ( Nelms and Goodnow 2001 ). Several large ENU screening programs have been established ( Justice et al. 1999 ).

Figure i

Boilerplate daily census of mouse cages in the centralized animal facilities at the Academy of Washington from 1993 to 2001. The population increased 161.3% over the period, an average of 23% per year.

All of the types of animals described in a higher place are necessary to produce the genetically modified lines that must then be bred to produce animals to study. They practise not, in themselves, produce usable research data. The number of animals not producing data are increased past the small percentage of offspring produced past pronuclear injection, which express the desired genetic alterations in addition to those used in creating crossbred and backcrossed lines of targeted mutations. Even after a line is created, nontransgenic and wild-type littermates may be produced that are not suitable for enquiry or farther convenance. Euthanasia is a common fate for these animals.

Some other aspect of the numbers issue stems from the fact that once a line is established, it may be necessary to continue breeding animals that have weather condition producing welfare bug. Simply to maintain a line, mice with compromised health may exist bred but not studied; and then these animals also do non produce information straight. A solution for this situation may be the use of embryo or sperm cryopreservation to maintain lines that are non being actively studied ( Agca 2000 ; Critser and Mobraaten 2000 ; Rall et al. 2000 ).

The welfare issue of an increasing number of genetically engineered animals may exist offset by the pursuit of refinement, another one of the 3Rs of animal use. Genetically altered animals are proving to be more exact or precise models of disease than many of the spontaneous and experimentally induced models used in the past. They may ultimately reduce the number of animals needed for enquiry by producing more than authentic data. Already, promising results are beingness reported in creating genetically engineered models for diseases in which enquiry has previously been hampered past a lack of reliable models. Examples are discussed below.

When studies involving newly created lines of genetic engineered animals or established lines that are new to an institution are proposed, the IACUC, enquiry team, caretakers, and veterinary staff all are faced with the need to ascertain any special requirements for maintaining the wellness and well-being of the animals. The process of determining the requirements for newly created lines may exist more complicated and hard than for previously characterized ones. The first questions to exist asked should business organization what is known about the lines and what are the expected outcomes. The sources of information to answer the questions will vary with the situation.

Studies of Documented Lines

A series of databases can be searched by an investigator or an IACUC reviewer to ascertain what is known regarding lines of genetically engineered animals that take been characterized previously. The periodical Nucleic Acids Research annually updates and publishes a list of molecular biology databases ( Baxevanis 2001 ). In 2001, the listing included 281 databases, some of which provide information regarding cistron expression in a multifariousness of species used in genetic applied science studies including mice, Xenopus (frogs), and zebrafish.

Ane of the most useful databases for investigators and IACUC members is the transgenic animal/targeted mutation database TBASE (<http://tbase.jax.org>) ( Woychik et al. 1993 ). It provides data well-nigh lines of transgenic and targeted mutant mice. Each entry includes the proper noun of the line and method used to generate it (i.e., pronuclear injection, homologus recombination), DNA construct used, genetic groundwork of host embryo or stem cells, phenotype associated with expression of the genetic change, effects of crossing the line with other mutant lines, how the line is maintained, author's comments, and a contact person to arrange for acquisition of the animals. TBASE also reports the age of onset of changes in phenotype produced by the mutation, progression of disease atmospheric condition, and points at which euthanasia should be considered. It can be a source of data about ways to care for animals to prevent or ameliorate progression of signs.

Tabular array one contains useful information about selected lines discussed in this article. As an example of how the database is helpful in the identification of welfare concerns, an investigator proposed a study using cystic fibrosis transmembrane regulator gene knockout mice (CTFR-/-) of the line designated the S489X mutation. A check of the TBASE site revealed that there were four CTFR (-/-) lines (ID nos. 1094, 1113, 1236, and 3436). Ane of the lines (ID no. 1113) was designated as the S489X mutation. The line was produced past homologous recombination in E14TG2a ES cells and injection of C57BL/6 blastocysts. The heterozygous phenotype was described as wild-blazon, meaning information technology was unaltered. The homozygous phenotype was reported to be altered, with postnatal lethality. Many deaths occur during the start 5 postnatal days, and very few mice live beyond 30 days. The homozygous mutants are runted and have severe intestinal obstruction leading to death by peritonitis. Because heterozygous animals are phenotypically normal, it is possible to utilize them for maintaining the line and to produce only the number of affected homozygous animals necessary to achieve the proposed studies. The reports are not exhaustive and may be dated, and so the database should be augmented by information gathered in a current search of the literature. Using a search, the investigator learned that the intestinal obstruction bug in CTFR (-/-) mice tin be alleviated by feeding pups a low-residue liquid diet outset at 10 days of age ( Eckman et al. 1995 ). This instance illustrates that TBASE and a search of the literature tin can be useful initial indicators of the utility of mutant lines and of welfare problems that should be addressed if they are to be used.

Table 1

Examples of welfare issues reported in the TBASE database for selected transgenic and knockout lines of mice a

Line proper noun b	(+/-) b	(-/-)	Lethality	Phenotype (clinical abnormalities)
ApoE (-/-)	A	A	No	Atherosclerosis on high-fat diet, elevated LDL and VLDL, no disease signs on low-fat diet
CFTR (-/-) S489X	WT	A	Postnatal	Runting at birth, almost died by 1 mo. Weight loss, abdominal distention, intestinal obstacle and rupture, gallbladder distention and rupture
LDLR (-/-)	A	A	No	Elevated LDL cholesterol levels, no illness signs noted
LIF (-/-)	WT	A	No	Retarded growth, female homozygous -/- are infertile.
RAG-i (-/-)	WT	A	No	No B or T lymphocytes, no IgM, b appear normal to 21 wk, small-scale size
Tro53 (-/-)	A	A	No	Develop multiple tumors (lymphomas and sarcomas) by half-dozen mo
SOD/G93A	A		Postnatal	Hind limb weakness and paralysis, moribund past five mo

Line proper noun b	(+/-) b	(-/-)	Lethality	Phenotype (clinical abnormalities)
ApoE (-/-)	A	A	No	Atherosclerosis on high-fatty diet, elevated LDL and VLDL, no disease signs on depression-fat diet
CFTR (-/-) S489X	WT	A	Postnatal	Runting at birth, most died by one mo. Weight loss, abdominal distention, abdominal obstruction and rupture, gallbladder distention and rupture
LDLR (-/-)	A	A	No	Elevated LDL cholesterol levels, no disease signs noted
LIF (-/-)	WT	A	No	Retarded growth, female person homozygous -/- are infertile.
RAG-1 (-/-)	WT	A	No	No B or T lymphocytes, no IgM, b announced normal to 21 wk, small size
Tro53 (-/-)	A	A	No	Develop multiple tumors (lymphomas and sarcomas) past six mo
SOD/G93A	A		Postnatal	Hind limb weakness and paralysis, moribund by 5 mo

The phenotype, whether wild-blazon (WT) or altered (A), is stated for heterozygous (+/-) and homozygous (-/-) mutants. The caste of alteration of various parameters is described in the phenotype section of TBASE for each line. The alteration is often greater in (-/-) than (+/-).

A, altered; ApoE, apolipoprotein E; CFTR, cystic fibrosis transmembrane regulator; IgM, immunoglobulin Yard; LDLR, low-density lipoprotein receptor; LIF, leukemia inhibitory factor; RAG, recombination-activating gene; Trp, transforming-related poly peptide; WT, wild-type.

Table 1

Examples of welfare bug reported in the TBASE database for selected transgenic and knockout lines of mice a

Line name b	(+/-) b	(-/-)	Lethality	Phenotype (clinical abnormalities)
ApoE (-/-)	A	A	No	Atherosclerosis on loftier-fat nutrition, elevated LDL and VLDL, no affliction signs on low-fatty nutrition
CFTR (-/-) S489X	WT	A	Postnatal	Runting at nascence, well-nigh died by i mo. Weight loss, abdominal distention, intestinal obstruction and rupture, gallbladder distention and rupture
LDLR (-/-)	A	A	No	Elevated LDL cholesterol levels, no disease signs noted
LIF (-/-)	WT	A	No	Retarded growth, female homozygous -/- are infertile.
RAG-1 (-/-)	WT	A	No	No B or T lymphocytes, no IgM, b announced normal to 21 wk, small-scale size
Tro53 (-/-)	A	A	No	Develop multiple tumors (lymphomas and sarcomas) by half dozen mo
SOD/G93A	A		Postnatal	Hind limb weakness and paralysis, moribund by 5 mo

Line proper name b	(+/-) b	(-/-)	Lethality	Phenotype (clinical abnormalities)
ApoE (-/-)	A	A	No	Atherosclerosis on high-fat nutrition, elevated LDL and VLDL, no disease signs on low-fat diet
CFTR (-/-) S489X	WT	A	Postnatal	Runting at birth, most died by one mo. Weight loss, abdominal distention, intestinal obstruction and rupture, gallbladder distention and rupture
LDLR (-/-)	A	A	No	Elevated LDL cholesterol levels, no disease signs noted
LIF (-/-)	WT	A	No	Retarded growth, female person homozygous -/- are infertile.
RAG-1 (-/-)	WT	A	No	No B or T lymphocytes, no IgM, b appear normal to 21 wk, small size
Tro53 (-/-)	A	A	No	Develop multiple tumors (lymphomas and sarcomas) past six mo
SOD/G93A	A		Postnatal	Hind limb weakness and paralysis, moribund by 5 mo

The phenotype, whether wild-type (WT) or altered (A), is stated for heterozygous (+/-) and homozygous (-/-) mutants. The degree of alteration of various parameters is described in the phenotype section of TBASE for each line. The alteration is often greater in (-/-) than (+/-).

A, altered; ApoE, apolipoprotein E; CFTR, cystic fibrosis transmembrane regulator; IgM, immunoglobulin M; LDLR, low-density lipoprotein receptor; LIF, leukemia inhibitory factor; RAG, recombination-activating factor; Trp, transforming-related protein; WT, wild-type.

Another investigator proposed using mice with the Trp53 tumor suppressor factor knocked out. A check of TBASE revealed numerous lines on several unlike background strains. It was learned that they all have an increased incidence of tumors, with the type of tumor and historic period of onset dependent on the background strain of mouse. On a 129/Sv background (at present 129/P), the most often observed tumor is malignant lymphoma, and testicular tumors are the next about mutual. The age of the animals at the time of onset of the tumors is 5 wk, and all develop tumors by 6 mo. More than half of the lymphomas involve the thymus and lead to respiratory distress and requiring euthanasia. Like p53 knockout mice on a background that is 75% C57BL/6 and 25% 129/Sv besides develop malignant lymphomas as the predominant tumor. On this background, the age of onset is slightly older, with 74% afflicted by 6 mo and all affected by 10 mo of age. Testicular tumors are not commonly seen on this background. With this knowledge, it is possible to devise precise monitoring protocols and schedules for each line and to define endpoints.

Other relevant databases available through the Mouse Genome Informatics web site (<http://www.informatics.jax.org>) include the Mouse Genome Database ( Blake et al. 2001 ), the Mouse Gene Expression Database ( Ringwald et al. 2001 ), and the Mouse Tumor Biological science database ( Bult et al. 2001 ). An investigator or reviewer who uses these resources can access information regarding lines that have been characterized to define problems that accept been experienced.

Studies of New and Uncharacterized Lines

Many of the welfare problems in newly created lines of transgenic animals occur unexpectedly. It is difficult to predict problems because outcomes can vary in different lines produced using the same methodology and DNA constructs due to the randomness of the process of incorporating the DNA construct into the genome. With transgenic mice, the sites of incorporation tin can vary when one attempts to produce similar lines of animals using identical DNA constructs. A line with i copy of the Deoxyribonucleic acid construct may have a different phenotype from ane with two or more copies. In add-on, a line with one construct of Dna incorporated into a particular chromosome tin have a different phenotype from another line with a copy of an identical construct of Dna incorporated into a different chromosome or even a different location on the same chromosome. Variations in regulatory sequences activated or inactivated, and differences in the background strain of mouse (seen with Tg, ENU, and knockouts) are among other influences that tin can produce phenotypic dissimilarities in genetically contradistinct mouse lines.

Palmiter and colleagues (1982) illustrated the variability of phenotypes of transgenic mice early on in the history of transgenic mouse product past dramatically demonstrating the power to produce a very big mouse by microinjection of the gene for rat growth hormone into mouse embryos. The size increase that occurred in the transgenic offspring is the phenotypic expression that ane would predict as the outcome from overexpression of the growth hormone factor. Unfortunately, subsequent attempts to produce similar lines resulted in lines with a diversity of phenotype issues, including liver and kidney failure, increased tumor production, and shortened life span ( Wolf and Wanke 1995 ). These unexpected problems illustrate the difficulty in predicting outcomes when creating new lines of transgenic animals. Examples of similar types of unanticipated outcomes in other lines are described elsewhere in the literature ( Dennis 1999 , 2000 ). This randomness of incorporation is not a problem in genetically modified lines produced by other methods. However, at that place is likewise a big variation in the phenotypes produced in ENU mutagenesis due to the randomness or variation in which particular genes are mutated.

In newly created genetically altered lines, an effective fashion to accost welfare problems is through a system with two interdependent components. One component is the surveillance for clinical problems by the animal intendance, research, and veterinarian staffs; the other component is the assessment of the phenotype by the research team.

Clinical Surveillance

A surveillance system should include frequent ascertainment of animals to identify concerns early and a reporting system for veterinary evaluation of morbidity and mortality. The program of adequate veterinary care should endeavor to detect and appraise affliction, physical arrears, injury, or abnormal behavior. In genetic engineering science studies, animals should be observed at least daily and mayhap more oftentimes if uncharacterized lines with issues are being produced. Surveillance is a duty that the animal intendance, research, and veterinary staffs should share. Newly created lines tin present a particularly challenging problem in ensuring that the system is acceptable to detect the wide variety of conditions that tin occur. Some genetic alterations may produce unexpected syndromes never encountered before. Even seemingly insignificant changes should be studied and documented as potential effects of the genetic amending. The surveillance program should emphasize vigilance for bear witness of annihilation abnormal.

Although the phenotype changes produced in a particular newly created line may exist challenging to predict, every bit soon every bit a detail line has been characterized, the types of clinical issues encountered and their time of onset are often more than uniform than encountered in their wild-type ancestors. This situation can be helpful in designing monitoring protocols for established lines. A surveillance plan should be tailored to ensure that it will identify and accost the issues that have been reported in the literature or databases to touch the line. Often the near important parameters to monitor in a item line and the crucial time points for increased frequency of observation tin exist identified. Still, there is enough variation that regular examination and vigilance for unforeseen problems is as important as it is for animals in other types of studies. At the University of Washington, the most frequently encountered unanticipated problems are infectious in nature; however, noninfectious problems accept also occurred. One example involved apolipoprotein E knockout mice (ApoE -/-) on a C57BL/6 background. A database review revealed that these severely hypercholesterolemic mice develop lesions of atherosclerosis but experience no outward signs of disease. However, there is no mention that older animals are consistently establish to accept thickened, ulcerated pare, with intense pruritis and xanthomas, which are consistent findings in our colony. This case is ane of several that illustrate the necessity of monitoring for any atmospheric condition that may ascend, non only for those already identified.

It is of import for a surveillance system to include regular evaluation for murine pathogens and to consider the use of serology, necropsy, and histology. If immune-deficient lines incapable of antibody production are being used, a sentinel programme may be necessary to detect the presence of pathogens ( Rehg and Toth 1998 ). In addition, immune-deficient mutant animals may exist affected by diseases not commonly seen as clinical problems with immune-competent animals. Inflammatory bowel affliction has been reported in both athymic (nude) and severe combined allowed-deficient mice ( Ward et al. 1996 ). Rag1-/- and interleukin (IL)-10-/- mice take also been shown to be susceptible to experimentally induced infection with severity dependent on the background strain ( Burich et al. 2001 ). Pneumonia due to Pneumocystis carinii has been found in mice with the T cell receptor alpha or beta knocked out; whereas, wild-type animals on the same background strain take been resistant. It is worth noting that infectious problems are not commonly noted on TBASE.

Phenotype Assessment

When a new mutant line is created, it is the responsibleness of the research squad to assess the phenotypic differences between the mutant animals and the wild-type. Of the many protocols proposed, virtually employ observation and minimally invasive tests for general, broad-range characterization of a line. A careful necropsy, including histology when gross lesions are found, should be a component of any basic phenotyping protocol. This is particularly important for animals that are observed to be ill or die unexpectedly. It is possible to follow these methods with more specialized testing to appraise particular abnormalities or to assess the potential utility of the mutant line for a particular research surface area ( Becker et al. 1996 ; Crawley 1999 ; Rogers et al. 1997 ; Wood 2000 ). Behavior phenotyping is an example of more specialized testing, which is existence used with increasing frequency. The results of phenotype testing should be available to the IACUC to review when an investigator requests approval for continued breeding of a line.

Virus Vectors

Both virus and nonvirus vectors are under investigation as carriers of genetic material for gene therapy for both humans and animals. Intact virus is not used to comport strange genetic material due to concerns that the vector might produce an infection when injected, or that the transgene might be carried into other animals or humans through horizontal transfer. Business concern virtually infection stems from an incident in which the Moloney murine leukemia virus was used every bit a gene therapy vector in rhesus monkeys later on whole trunk irradiation. Employ of the replication-competent virus was believed to be safe because information technology is not known to exist a pathogen for primates. 3 of 8 recipients of the gene therapy developed a T cell lymphoma within seven mo of receiving the virus ( Donahue et al. 1992 ). Although it is not conclusive, information technology must be suspected that the irradiation contradistinct the monkeys' susceptibility to the virus vector. Due to concerns almost such outcomes, virus vectors for gene therapy are normally rendered replication deficient. Nevertheless, even use of replication-defective viruses is accompanied by concern that they could cause welfare issues in recipient animals. If a helper virus were available, the vector virus might regain the ability to replicate and transmit the transgene, unacceptably, to other animals or humans. Another theoretical trouble that must be considered is the outcome of an underlying disease on the host's susceptibility to the virus vector.

Ensuring Welfare

Containment

One common aim of studies of genetically engineered animals is to learn the consequences of the presence, absence, or alteration of a particular gene. Compromised welfare is usually not intended; however, in that location are issues that demand attention when animals with an altered genome are used in inquiry and testing. Even if the alterations produce no change in the phenotype of the animals, the welfare of feral populations and the environment must be considered. If animals whose genome has been altered past the stable introduction of recombinant DNA into the germ line should escape and breed with feral populations, the environment could be contradistinct and a disastrous situation might be created. To forestall this possible event, altered animals must, therefore, be contained under BL1-N weather ( Federal Register 1994 ). This degree of containment involves standard microbiological (BL1) practices and limited access to the laboratory when experiments are in progress. Institutions are obligated to take containment measures to prevent the escape of genetically modified animals and establish programs to prevent feral rodents from gaining access to the animal facilities. Unless breeding or reproductive studies are office of the experiment, a bulwark should be provided to separate males and females. As an added containment measure, live mice that are genetically contradistinct should not be released to zoos or pet stores to exist used for animal food. To identify animals to be contained, the Guidelines for Research Involving Recombinant Dna ( Federal Register 1994 ) require the permanent marker of genetically engineered animals larger than rodents inside 72 hr of birth. If their size does non permit permanent marking, their container should be marked.

The occurrence of allowed deficiency is also a potential source of welfare problems that should be addressed by containment. Alterations of histocompatibility and regulatory genes or inactivation of genes required for a particular allowed part are among many causes of compromise of immune part in genetically engineered animals. It is also common to breed a particular mutation onto a astringent combined immune-scarce (SCID) or athymic (nude) strain of mice. Immune-deficient animals crave special living conditions to protect them from organisms that may non be pathogenic to their immune-competent siblings. It is helpful to house immune-compromised animals in ventilated cages with filtered air and to provide sterile cages, bedding, food, and water to prevent occurrence of fatal septicemia. The use of filtered air change stations and protective clothing is too helpful. Even with these precautions, animals may go infected. Cesarian rederivation or continuous antimicrobial therapy may be necessary to control these conditions.

Handling

When welfare problems are encountered in lines of genetically engineered animals, treatment is i option that should exist considered. An case of successful utilize of this strategy is the administration of Fifty-DOPA to mice defective the tyrosine hydroxylase gene in dopaminergic neurones ( Zhou and Palmiter 1995 ; Zhou et al. 1995 ). Embryonic and neonatal death is the fate of the altered mice unless L-DOPA is administered. Treatment strategies have been used successfully in other disease models for many years. One common example is the administration of insulin to animals with type 1 diabetes mellitus.

Limitation of Expression

The power to limit gene expression to certain tissues is a method that has been used to study factor action in both normal and illness situations. A genetic change that would cause serious problems or expiry when expressed in all tissues tin exist limited to certain tissues of interest. In this situation, improved welfare is not the master reason for using the procedure, only information technology is an incidental benefit. The absenteeism of expression of the contradistinct genome in the other tissues may protect an brute from phenotype changes that would create welfare problems. One such method of limiting expression is the Cre- loxP recombination system ( Ray et al. 2000 ). Cre is an enzyme that catalyzes the removal of a DNA segment that lies between 2 specific 34 base-pair sequences, termed loxP . The system involves creation of two separate lines of mice. In i line, the gene to be knocked out is bracketed with two loxP sequences by homologous recombination. The other line has Cre sequences targeted to tissue specific promoters inserted into the genome of the cells of interest. The ii lines are then crossbred to produce a line that has the gene sequence excised in the cells of interest. The gene sequence remains nowadays and functional in the other tissues, although it continues to exist bracketed by ii loxP sequences.

The use of inducible promoters is another method of limiting gene action by turning them on and off for specific fourth dimension periods. Manifestation of a genetic alteration is needed only for the period of time necessary to accomplish a study. When these techniques are used, genetic manipulations that would potentially result in welfare problems or even decease of the animals can now be accomplished and studied. Past using a tetracycline promoter ( Furth et al. 1994 ), a gene of interest tin be normal while the creature does non receive tetracycline. Gene part can be stopped by feeding the animal tetracycline and restored past discontinuing assistants of the tetracycline. Using this system, the animal can have a normal phenotype before and afterward the written report. In improver, Kistner and colleagues (1996) have described a reverse tetracycline organisation using a doxycycline-inducible promoter. An inserted factor sequence is activated by adding doxycycline to the drinking h2o. When the doxycycline is stopped, the inserted sequence becomes inactive. Other systems that allow the controlled expression of transgenes include the ecdysone-inducible system ( No et al. 1996 ), the CYP1A1 promoter ( Campbell et al. 1996 ), and a metallothionein promoter ( Choo et al. 1986 ). These systems can even be used to written report embryonically lethal conditions ( Sarao and Dumont 1998 ).

Establishing Endpoints

Humane endpoints for animals used in research and testing are discussed in a recent outcome of ILAR Periodical ( Carstens and Moberg 2000 ; Dennis 2000 ; Hendriksen and Steen 2000 ; Morton 2000 ; Olfert and Godson 2000 ; Sass 2000 ; Stokes 2000 ; Toth 2000 ; Wallace 2000 ). In the design phase of a study, consideration should exist given to the signal at which an animal should be removed from the study. Many genetically manipulated lines lend themselves to the utilize of objective endpoints because of relative consistency in the occurrence of the conditions that announced, the time bespeak at which they appear, and their severity. Scoring systems have been used with success in studies involving lines showing this consistency. There are many examples of innovative scoring systems, and some have fifty-fifty been automatic for hand-held computers ( Hampshire 2001 ). Some systems, such as body condition scoring, utilise consistent criteria that can apply to many unlike situations ( Ullman-Cullere and Folz 1999 ). Other systems may require adjustments to fit individual situations. Ane instance of such alteration involves monitoring of the extent of arthritis in transgenic mice using a score sheet with increasing point values assigned as signs of pain increased in severity. Several animals were observed to show signs of pain earlier the time when their point totals indicated they should be removed from the report. The problem was resolved past observing that when each individual bespeak value on the sail was squared, the total score provided a predictable and timely indication of when euthanasia should be considered ( Cheunsuk et al. 1999 ). With any scoring system, there must as well exist vigilance for unforeseen problems that may non fit the parameters designated for evaluation.

IACUC Oversight of Welfare Issues

The IACUC is responsible for reviewing and blessing proposed animate being studies. Every bit investigators create and learn new genetically altered animals, IACUC members are faced with reviewing proposals that tin can involve lines with a variety of problems such equally premature lethality, altered bodily functions, increased tumor production, decreased disease resistance, altered susceptibility to microorganisms, and many others. In the Guide for the Care and Use of Laboratory Animals , 1 of the topics suggested for review of brute care and apply protocols is "Criteria and process for timely intervention, removal of animals from a written report, or euthanasia if painful or stressful outcomes are anticipated" ( NRC 1996 , p. 10). It is customary for the IACUC to ask investigators to listing anticipated effects of the proposed manipulations and to describe the planned monitoring protocol for identifying bug.

As discussed above, when new lines are being developed, information technology may be hard to predict outcomes accurately. However, in that location is value in anticipating the possible problems even though the list may require revision every bit the written report develops. A vigilant surveillance system tin be helpful in identifying unpredicted events. The endpoints to exist used for euthanasia of affected animals should also be listed. It is oft possible to select endpoints that are relatively early in a disease process considering death is seldom necessary to document the effect or change of a particular genetic change. When problems are encountered, the attending veterinarian and the investigative team should piece of work together to identify ways to prevent or alleviate them. As new methods of limiting the timing and extent of expression of genetic changes are developed, investigators will employ them to create more precise and sophisticated models. As has been seen, these refinement methods can also preclude or convalesce many welfare problems.

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Abbreviations used in this article: ALS, amyotrophic lateral sclerosis; ENU, North-ethyl-N-nitrosourea; ES, embryonic stem; IACUC, institutional animal care and use committee; p53, short term for Trp53; Trp53, transforming-related poly peptide 53.

Source: https://academic.oup.com/ilarjournal/article/43/2/100/646403

Posted by: johnsonsniters.blogspot.com

What Are Some Of The Issues That Genetically Modified Animals Come Across?

Abstract

Introduction

Types of Inquiry

Gene Discovery

Disease Models

Exam Arrangement Development

Gene Therapy

Xenotransplantation

Life Span Extension

Identification of Welfare Concerns

Welfare Issues in Production and Breeding

Studies of Documented Lines

Studies of New and Uncharacterized Lines

Clinical Surveillance

Phenotype Assessment

Virus Vectors

Ensuring Welfare

Containment

Handling

Limitation of Expression

Establishing Endpoints

IACUC Oversight of Welfare Issues

References

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