Genetically Modified Animals: Should There Be Limits To Engineering The Animal Kingdom?

Abstract

Genetically engineered animals accept opened new frontiers in the study of physiology and affliction processes. Mutant animals offer more accurate disease models and increased precision for pathogenesis and treatment studies. Their use offers hope for improved therapy to patients with conditions that currently have poor or ineffective treatments. These advantages have fostered an increase in studies using mice in recent years, a evolution viewed with alarm by those who oppose the utilize of animals in research. Scientists point out that the mice are replacing more sentient species, such as nonhuman primates, and are increasing the quality of enquiry beingness conducted. They assert that report of genetically engineered animals volition somewhen permit decreases in numbers of animals used in enquiry. Withal, the increment in use of genetically altered animals presents many challenges in reviewing protocols and providing care. Identification and resolution of any welfare problems is a responsibility that is shared past institutional brute care and apply committee, veterinary, animate being care, and research staffs. To place potential welfare concerns, a database such every bit TBASE (<http://tbase.jax.org>) can exist searched to learn what has been reported for established mutant lines. In add-on, newly created lines should exist monitored by a surveillance system and have phenotype assessment to place the effects of altering the genome. Methods of ensuring welfare can include treatment of weather condition produced, restriction of gene expression to tissues of involvement or to sure time periods, and establishment of endpoints for removing animals from a study earlier bug announced.

Introduction

Modification of the genome of animals has occurred throughout the ages. Initially, changes in genetic limerick occurred spontaneously. With the beginning of agriculture, humans exerted influence on the process by selecting animals with desirable genetic traits. An animal with a spontaneous genetic mutation that increased feed conversion, additional milk product, or produced more than desirable carcass characteristics would be selected as convenance stock to ameliorate the herd by perpetuating advantageous phenotype changes. More than recently, scientists accelerated the mutation process with irradiation and chemical 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 evolution that resulted from the injection of strange Deoxyribonucleic acid into the pronucleus of embryos ( Gordon et al. 1980 ; Palmiter et al. 1982 ). Offspring expressed the injected DNA construct and passed information technology on to succeeding generations. The ability to dispense and study the genome has increased greatly with the subsequent development of cistron targeting technology, which allows an investigator to knock out a gene sequence of interest in embryonic stem (ES one ) cells ( Capecchi 1989 ). Signal mutagenesis accomplished past administration of N-ethyl-N-nitrosourea (ENU 1 ) is another tool in widespread use to create new illness models.

This article discusses some of the areas of research that are aided by the creation of these animals, welfare issues that have been associated with their production, sources of information to help predict bug, tools available to forestall or limit any welfare difficulties that occur, issues in institutional animal intendance and utilise committee (IACUC 1 ) review of studies of genetically altered animals, and strategies for addressing welfare issues. Give-and-take of animal welfare issues 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 offer more than accurate models that provide more precise data than previously used paradigms, permit the replacement of higher species with less sentient species, and may ultimately reduce the number of animals required to address a particular disease problem. These potential advantages are examined throughout this discussion.

Types of Research

The mapping of the human genome is stimulating i of the most exciting periods in the history of biomedical enquiry. Studies are in progress that could develop new ways to treat disease conditions past altering the genome. The prospect of such treatments has aroused controversy regarding the ethics of potential treatments that would alter the human 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 be limited to modalities that alter genes only in somatic cells. These alterations would not touch offspring of treated patients.

In the near future, germline-modifying therapies will probably be express to laboratory animals. The ability to manipulate the genome of animals makes them an indispensable component of inquiry involving amending and modification of genes. It is expected that the research will produce unprecedented breakthroughs in medicine, merely a few of the genetically altered animals created may nowadays challenges to the IACUC, the veterinarian staff, and others striving to ensure animal well beingness. Among the areas studied are gene discovery, illness models, test systems, cistron therapy, xenotransplantation, and life span extension.

Cistron Discovery

Gene discovery is an area of investigation designed to determine the structure and part of various genes. A closely related area is proteomics, in which proteins produced past genes are studied to find their action and the interactions with other proteins in the body. Some of these studies are genome driven and apply lines of mice created with man or fauna genes of interest beingness either expressed or inactivated and determining the resultant phenotype. Other studies are phenotype driven and apply mutagens such every bit ENU and chlorambucil to produce animals with altered phenotypes. These animals are then studied to determine the cistron alterations that produced the detail phenotype.

Because many of the genes to exist studied are necessary for fetal development or the basic functions of life, some of the animals produced will likely accept health problems and increased lethality. Ensuring animal welfare in some of these newly created lines may require vigilant surveillance and innovative direction.

Disease Models

Application of genetic engineering techniques has enabled the cosmos of new models for human diseases that have previously lacked accurate spontaneous or experimentally induced animal models. Conditions such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS one ), and Parkinson's disease are subjects of intense efforts to develop new models. I 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 three to four mo of age that progresses to paralysis past 5 mo. The syndrome is like to that seen in human patients with ALS. Even research for diseases that previously had many models, such as diabetes mellitus, has been aided past new models developed using transgenic and knockout technology ( Wong et al. 1999 ). The evolution of improved models that offering advantages over existing ones volition continue and will improve the quality of inquiry. Because the lines created will manifest signs similar to those seen in humans affected by the same syndrome, they may nowadays challenging welfare issues.

Work is in progress to develop new models past altering the susceptibility of mice to pathogens of humans. Considering the but models for conditions such as viral hepatitis and AIDS have been in nonhuman primates, the utilise of genetically altered mice offers the potential creature welfare improvement of performing the research in a less sentient species. There is some concern that genetically contradistinct mice might provide a reservoir for these human pathogens and spread infection to the homo population. Even so, the hazard from the mice is non greater than from similarly affected nonhuman primates. Information technology is crucial to preclude escape of altered animals to preclude breeding with wild animate being populations.

Research 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 1 ) tumor suppressor cistron ( Donehower et al. 1995 ; Harvey et al. 1993 ; Purdie et al 1994 ). These lines feel an increased incidence of tumors compared with their wild-type relatives. The frequency, age of onset, and even tumor blazon varies with dissimilar background strains. Usually used strains for these models include C57BL/six and 129/P.

It is important to appraise the phenotype of each new line created to discover new illness models. An instance of the importance of examining each new line is illustrated by a particular strain, C57BL/6J-TgN(LckIL4)1315Dbl, of transgenic mice for interleukin-4 ( Lewis et al. 1993 ). In contrast to lines previously created with similar constructs, one line (no. 1315) became progressively humpbacked starting at iii to six mo of historic period. Phenotype assessment revealed that the animals were affected with osteoporosis. This unique line became a valuable model for studying the previously unrecognized role of interleukin-4 in osteoporosis.

Examination Arrangement Development

Genetic engineering science engineering is beingness 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 sentient species, such as nonhuman primates, in some of these assessments. For instance, transgenic mice expressing the human polio virus receptor have been created, and they show promise as substitutes for nonhuman primates to test the rubber of attenuated 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 exam systems with increased sensitivity for the detection of carcinogenic chemicals.

Every bit gene therapy modalities are developed, new condom testing strategies volition need to exist adult to assess the effects of the transgene being delivered as well equally any toxic effects of vector systems used for delivery. The US Food and Drug Assistants recognizes that fauna models created through genetic or pharmacological ways can be used in an endeavour to demonstrate that gene therapy products can correct a genetic defect, deadening progression of a disease, or alleviate its signs ( Pilaro and Serabian 1999 ).

Cistron Therapy

Treating disease conditions past altering the genome of somatic cells of affected humans and animals is an heady area of inquiry that provides promise for conditions that presently lack effective treatment modalities. Examples of changes beingness investigated include replacement of a defective factor with a normal 1, insertion of resistance genes, or altering regulatory sequences to turn genes on or off. Research in this area involves creation of laboratory animals with specific genetic defects so correcting the defects past therapy with normal genes. In reviewing these protocols, information technology is of import to consider the welfare issues of untreated control groups.

Xenotransplantation

The demand for transplantable organs is fostering inquiry into the creation of animals whose tissues are uniform for implanting into humans. Larger species, such as pigs and baboons, are preferred for development as donors because of the similarity of their organ size to that of humans. All the same, mice are beingness used in preliminary studies to assess feasibility and establish procedures to make the animate being tissues compatible. These studies might produce animals with modified immune systems that are susceptible to a diverseness of organisms, some of which are before long believed to be nonpathogenic for the species. These animals may present some novel diagnostic and therapeutic challenges.

Life Span Extension

As medical research has progressed, the advances in control of disease have resulted in a steady increase in longevity for humans, with increased health and vigor for the elderly. Contempo results with genetically altered animals are raising hopes that possibly life span tin be increased even farther. I 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 accept increased resistance to oxidative stress and a 30% increased life span compared with wild-type mice ( Migliaccio et al. 1999 ).

The sit-in of such genetic alterations that outcome in increases in life span is a powerful incentive for research into the genetic control of aspects of the crumbling procedure. This area of investigation will remain active in the about future. In this search for genes that will increase longevity, in that location will also be the cosmos of some lines with shorter life bridge and welfare problems.

Identification of Welfare Concerns

Welfare Issues in Production and Breeding

Ane of the nearly controversial and vexing problems associated with the employ of genetically engineered animals derives from the abrupt increase in numbers of mice used in recent years. Opponents of their use assert that any welfare gains offered by refinement and replacement of other models are first by the failure to reduce the total number of animals used. The increase in number of mice used in enquiry in recent years tin can be attributed to intensified efforts in areas that were previously hampered by a lack of adequate models, every bit well as the development of new technologies.

Although accurate estimates for the number of mice used in enquiry in the United states of america are not published, I believe that the expansion in mouse populations in centralized facilities at my institution over the past eight yr is consequent with what is occurring in similar institutions throughout the country. As illustrated in Figure 1 , the increase is a total of 161.iii% or a charge per unit of more 23% per twelvemonth. The increment is due not just to growth in the numbers of animals on studies but likewise to the large number of animals necessary to create each genetically modified line. In transgenic and knockout lines, these animals include convenance 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 breed with females to produce pseudopregnant recipients for the altered embryos. In point mutagenesis studies, males are administered ENU to induce mutations in their spermatogonial stem cells. Approximately one in 700 gametes will take a mutation at any given locus, with multiple contained mutations per offspring obtained. Depending on the screening protocol, <one% to up to ten% of animals will have interesting phenotypes ( Balling 2001 ; Nelms and Goodnow 2001 ). Upwardly to three generations of convenance and screening may exist required to detect 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 1

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

All of the types of animals described above are necessary to produce the genetically modified lines that must then be bred to produce animals to study. They practice not, in themselves, produce usable research data. The number of animals not producing data are increased by the minor percentage of offspring produced past pronuclear injection, which limited the desired genetic alterations in add-on to those used in creating crossbred and backcrossed lines of targeted mutations. Fifty-fifty later a line is created, nontransgenic and wild-blazon littermates may exist produced that are not suitable for enquiry or further convenance. Euthanasia is a common fate for these animals.

Another aspect of the numbers consequence stems from the fact that once a line is established, it may exist necessary to proceed breeding animals that have weather condition producing welfare problems. Simply to maintain a line, mice with compromised health may be bred just non studied; so these animals likewise do not produce information straight. A solution for this state of affairs may be the employ of embryo or sperm cryopreservation to maintain lines that are not beingness actively studied ( Agca 2000 ; Critser and Mobraaten 2000 ; Rall et al. 2000 ).

The welfare issue of an increasing number of genetically engineered animals may be offset past the pursuit of refinement, another ane of the 3Rs of animal use. Genetically altered animals are proving to be more than verbal 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 research by producing more accurate data. Already, promising results are being reported in creating genetically engineered models for diseases in which research has previously been hampered by 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 establishment are proposed, the IACUC, enquiry squad, caretakers, and veterinary staff all are faced with the demand to ascertain any special requirements for maintaining the health and well-being of the animals. The procedure of determining the requirements for newly created lines may be more complicated and difficult than for previously characterized ones. The first questions to exist asked should concern what is known about the lines and what are the expected outcomes. The sources of data to answer the questions will vary with the situation.

Studies of Documented Lines

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

One of the most useful databases for investigators and IACUC members is the transgenic beast/targeted mutation database TBASE (<http://tbase.jax.org>) ( Woychik et al. 1993 ). It provides information about lines of transgenic and targeted mutant mice. Each entry includes the name of the line and method used to generate it (i.e., pronuclear injection, homologus recombination), DNA construct used, genetic background of host embryo or stalk cells, phenotype associated with expression of the genetic change, furnishings 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 likewise reports the age of onset of changes in phenotype produced by the mutation, progression of disease conditions, and points at which euthanasia should be considered. It can be a source of information about ways to care for animals to prevent or ameliorate progression of signs.

Table i contains useful data 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 cistron 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). One of the lines (ID no. 1113) was designated as the S489X mutation. The line was produced by homologous recombination in E14TG2a ES cells and injection of C57BL/6 blastocysts. The heterozygous phenotype was described as wild-type, significant it was unaltered. The homozygous phenotype was reported to be altered, with postnatal lethality. Many deaths occur during the outset 5 postnatal days, and very few mice live beyond 30 days. The homozygous mutants are runted and take severe abdominal 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 merely the number of affected homozygous animals necessary to reach the proposed studies. The reports are non exhaustive and may be dated, so the database should exist augmented by information gathered in a electric current search of the literature. Using a search, the investigator learned that the intestinal obstruction problems in CTFR (-/-) mice can be alleviated by feeding pups a low-residue liquid diet beginning at 10 days of age ( Eckman et al. 1995 ). This example illustrates that TBASE and a search of the literature 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 problems reported in the TBASE database for selected transgenic and knockout lines of mice a

Line proper name b	(+/-) b	(-/-)	Lethality	Phenotype (clinical abnormalities)
ApoE (-/-)	A	A	No	Atherosclerosis on high-fat diet, elevated LDL and VLDL, no illness signs on low-fat diet
CFTR (-/-) S489X	WT	A	Postnatal	Runting at birth, most died by 1 mo. Weight loss, abdominal distention, intestinal obstruction and rupture, gallbladder distention and rupture
LDLR (-/-)	A	A	No	Elevated LDL cholesterol levels, no affliction 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 appear normal to 21 wk, small size
Tro53 (-/-)	A	A	No	Develop multiple tumors (lymphomas and sarcomas) by 6 mo
SOD/G93A	A		Postnatal	Hind limb weakness and paralysis, moribund past 5 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 low-fat diet
CFTR (-/-) S489X	WT	A	Postnatal	Runting at birth, most died by 1 mo. Weight loss, intestinal 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-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 by 5 mo

The phenotype, whether wild-blazon (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 oftentimes greater in (-/-) than (+/-).

A, altered; ApoE, apolipoprotein E; CFTR, cystic fibrosis transmembrane regulator; IgM, immunoglobulin One thousand; 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 problems 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 high-fatty nutrition, elevated LDL and VLDL, no affliction signs on depression-fat diet
CFTR (-/-) S489X	WT	A	Postnatal	Runting at birth, about died by 1 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-scale size
Tro53 (-/-)	A	A	No	Develop multiple tumors (lymphomas and sarcomas) by 6 mo
SOD/G93A	A		Postnatal	Hind limb weakness and paralysis, moribund by 5 mo

Line name 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 nascency, most died by ane mo. Weight loss, intestinal 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) by half dozen 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 caste of alteration of various parameters is described in the phenotype section of TBASE for each line. The amending is often greater in (-/-) than (+/-).

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

Another investigator proposed using mice with the Trp53 tumor suppressor cistron knocked out. A check of TBASE revealed numerous lines on several different background strains. It was learned that they all have an increased incidence of tumors, with the blazon of tumor and age of onset dependent on the groundwork strain of mouse. On a 129/Sv background (now 129/P), the virtually oft observed tumor is malignant lymphoma, and testicular tumors are the next most common. The age of the animals at the time of onset of the tumors is v wk, and all develop tumors by 6 mo. More half of the lymphomas involve the thymus and lead to respiratory distress and requiring euthanasia. Similar p53 knockout mice on a background that is 75% C57BL/6 and 25% 129/Sv also develop malignant lymphomas every bit the predominant tumor. On this background, the age of onset is slightly older, with 74% affected by 6 mo and all affected by 10 mo of historic period. Testicular tumors are not ordinarily seen on this background. With this knowledge, it is possible to devise precise monitoring protocols and schedules for each line and to ascertain endpoints.

Other relevant databases available through the Mouse Genome Informatics web site (<http://www.information science.jax.org>) include the Mouse Genome Database ( Blake et al. 2001 ), the Mouse Factor Expression Database ( Ringwald et al. 2001 ), and the Mouse Tumor Biological science database ( Bult et al. 2001 ). An investigator or reviewer who uses these resource tin can access information regarding lines that accept been characterized to ascertain bug that have 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 dissimilar lines produced using the same methodology and DNA constructs due to the randomness of the procedure of incorporating the DNA construct into the genome. With transgenic mice, the sites of incorporation can vary when 1 attempts to produce similar lines of animals using identical Dna constructs. A line with one re-create of the DNA construct may take a unlike phenotype from one with two or more copies. In addition, a line with one construct of Dna incorporated into a particular chromosome can have a different phenotype from another line with a copy of an identical construct of DNA incorporated into a different chromosome or even a unlike 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 amid other influences that can produce phenotypic dissimilarities in genetically altered mouse lines.

Palmiter and colleagues (1982) illustrated the variability of phenotypes of transgenic mice early in the history of transgenic mouse production by 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 one would predict equally the outcome from overexpression of the growth hormone gene. Unfortunately, subsequent attempts to produce similar lines resulted in lines with a variety of phenotype bug, including liver and kidney failure, increased tumor production, and shortened life span ( Wolf and Wanke 1995 ). These unexpected issues 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 past other methods. However, there is besides a large 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 constructive way to address welfare bug is through a system with two interdependent components. One component is the surveillance for clinical problems past the animal care, research, and veterinary staffs; the other component is the assessment of the phenotype past the research team.

Clinical Surveillance

A surveillance system should include frequent observation of animals to place concerns early and a reporting system for veterinary evaluation of morbidity and bloodshed. The program of adequate veterinary care should endeavor to detect and assess illness, physical arrears, injury, or abnormal behavior. In genetic engineering science studies, animals should exist observed at least daily and perhaps more than frequently if uncharacterized lines with problems are existence produced. Surveillance is a duty that the brute care, research, and veterinary staffs should share. Newly created lines tin can present a particularly challenging problem in ensuring that the system is acceptable to detect the wide multifariousness of conditions that tin can occur. Some genetic alterations may produce unexpected syndromes never encountered before. Even seemingly insignificant changes should be studied and documented every bit potential effects of the genetic alteration. The surveillance program should emphasize vigilance for testify of anything aberrant.

Although the phenotype changes produced in a detail newly created line may be challenging to predict, every bit soon as a particular line has been characterized, the types of clinical problems encountered and their fourth dimension of onset are often more uniform than encountered in their wild-type ancestors. This state of affairs can exist helpful in designing monitoring protocols for established lines. A surveillance program should be tailored to ensure that it will identify and address the problems that have been reported in the literature or databases to affect the line. Oft the most important parameters to monitor in a particular line and the crucial time points for increased frequency of observation can exist identified. Notwithstanding, there is enough variation that regular examination and vigilance for unforeseen problems is as important equally it is for animals in other types of studies. At the University of Washington, the nigh frequently encountered unanticipated problems are infectious in nature; all the same, noninfectious issues have too occurred. 1 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 simply experience no outward signs of disease. Yet, at that place is no mention that older animals are consistently constitute to take thickened, ulcerated pare, with intense pruritis and xanthomas, which are consistent findings in our colony. This case is 1 of several that illustrate the necessity of monitoring for any weather condition that may arise, non merely for those already identified.

It is important for a surveillance system to include regular evaluation for murine pathogens and to consider the use of serology, necropsy, and histology. If allowed-deficient lines incapable of antibiotic production are existence used, a lookout man program may exist 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 disease has been reported in both athymic (nude) and severe combined immune-deficient mice ( Ward et al. 1996 ). Rag1-/- and interleukin (IL)-x-/- 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 aforementioned background strain have been resistant. It is worth noting that infectious problems are non commonly noted on TBASE.

Phenotype Assessment

When a new mutant line is created, it is the responsibility of the enquiry team to assess the phenotypic differences between the mutant animals and the wild-blazon. Of the many protocols proposed, most use observation and minimally invasive tests for general, broad-range characterization of a line. A conscientious necropsy, including histology when gross lesions are found, should exist a component of any basic phenotyping protocol. This is specially of import for animals that are observed to be ill or die unexpectedly. It is possible to follow these methods with more than specialized testing to assess item abnormalities or to assess the potential utility of the mutant line for a detail research area ( Becker et al. 1996 ; Crawley 1999 ; Rogers et al. 1997 ; Wood 2000 ). Behavior phenotyping is an case of more specialized testing, which is being used with increasing frequency. The results of phenotype testing should exist available to the IACUC to review when an investigator requests approving 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 non used to carry foreign genetic fabric 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 most infection stems from an incident in which the Moloney murine leukemia virus was used as a gene therapy vector in rhesus monkeys after whole body irradiation. Use of the replication-competent virus was believed to be safe considering information technology is not known to be a pathogen for primates. Three of eight recipients of the gene therapy developed a T jail 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 altered the monkeys' susceptibility to the virus vector. Due to concerns about such outcomes, virus vectors for gene therapy are normally rendered replication deficient. Nonetheless, fifty-fifty utilize of replication-lacking viruses is accompanied by business organization that they could crusade welfare problems in recipient animals. If a helper virus were bachelor, the vector virus might regain the ability to replicate and transmit the transgene, unacceptably, to other animals or humans. Some other theoretical trouble that must be considered is the effect of an underlying illness on the host'southward susceptibility to the virus vector.

Ensuring Welfare

Containment

I common aim of studies of genetically engineered animals is to learn the consequences of the presence, absenteeism, or amending of a item gene. Compromised welfare is commonly not intended; however, there are bug that need attention when animals with an altered genome are used in inquiry and testing. Fifty-fifty if the alterations produce no alter in the phenotype of the animals, the welfare of feral populations and the environment must be considered. If animals whose genome has been altered by the stable introduction of recombinant Deoxyribonucleic acid into the germ line should escape and breed with feral populations, the environment could exist altered and a disastrous state of affairs might be created. To preclude this possible effect, altered animals must, therefore, exist contained under BL1-N conditions ( Federal Register 1994 ). This degree of containment involves standard microbiological (BL1) practices and express access to the laboratory when experiments are in progress. Institutions are obligated to have containment measures to prevent the escape of genetically modified animals and constitute programs to prevent feral rodents from gaining access to the fauna facilities. Unless breeding or reproductive studies are function of the experiment, a bulwark should exist provided to separate males and females. As an added containment measure, live mice that are genetically contradistinct should not exist released to zoos or pet stores to exist used for animate being food. To identify animals to be contained, the Guidelines for Research Involving Recombinant DNA ( Federal Register 1994 ) require the permanent marking of genetically engineered animals larger than rodents within 72 hr of birth. If their size does not permit permanent marker, their container should be marked.

The occurrence of immune 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 function are among many causes of compromise of immune function in genetically engineered animals. Information technology is also mutual to brood a item mutation onto a astringent combined immune-deficient (SCID) or athymic (nude) strain of mice. Immune-deficient animals require special living weather to protect them from organisms that may non be pathogenic to their allowed-competent siblings. It is helpful to house allowed-compromised animals in ventilated cages with filtered air and to provide sterile cages, bedding, food, and water to preclude occurrence of fatal septicemia. The use of filtered air change stations and protective article of clothing is besides helpful. Even with these precautions, animals may become infected. Cesarian rederivation or continuous antimicrobial therapy may be necessary to control these atmospheric condition.

Handling

When welfare problems are encountered in lines of genetically engineered animals, treatment is one option that should exist considered. An example of successful utilise of this strategy is the administration of L-DOPA to mice lacking the tyrosine hydroxylase cistron in dopaminergic neurones ( Zhou and Palmiter 1995 ; Zhou et al. 1995 ). Embryonic and neonatal death is the fate of the altered mice unless 50-DOPA is administered. Treatment strategies take been used successfully in other disease models for many years. One common instance is the administration of insulin to animals with blazon i diabetes mellitus.

Limitation of Expression

The ability to limit gene expression to certain tissues is a method that has been used to study factor action in both normal and disease situations. A genetic change that would crusade serious issues or decease when expressed in all tissues can exist limited to sure tissues of interest. In this situation, improved welfare is not the primary reason for using the procedure, but it is an incidental benefit. The absence of expression of the altered genome in the other tissues may protect an animal from phenotype changes that would create welfare issues. 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 ii specific 34 base of operations-pair sequences, termed loxP . The organization involves cosmos of two separate lines of mice. In one 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 two lines are so 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 be bracketed by two loxP sequences.

The utilise of inducible promoters is another method of limiting gene activeness by turning them on and off for specific time periods. Manifestation of a genetic alteration is needed only for the period of fourth dimension necessary to accomplish a written report. When these techniques are used, genetic manipulations that would potentially result in welfare problems or even death of the animals can at present be accomplished and studied. Past using a tetracycline promoter ( Furth et al. 1994 ), a gene of interest tin can be normal while the animal does not receive tetracycline. Cistron function can exist stopped by feeding the animal tetracycline and restored by discontinuing administration of the tetracycline. Using this system, the animal tin have a normal phenotype earlier and afterwards the study. In addition, Kistner and colleagues (1996) take described a contrary tetracycline arrangement using a doxycycline-inducible promoter. An inserted gene sequence is activated by adding doxycycline to the drinking water. 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 tin can even exist 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 contempo issue of ILAR Journal ( 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 stage of a study, consideration should be given to the betoken at which an beast should be removed from the study. Many genetically manipulated lines lend themselves to the use of objective endpoints because of relative consistency in the occurrence of the weather that appear, the fourth dimension point at which they announced, 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 even been automated for mitt-held computers ( Hampshire 2001 ). Some systems, such as body condition scoring, use consistent criteria that can apply to many different situations ( Ullman-Cullere and Folz 1999 ). Other systems may crave adjustments to fit individual situations. One example of such amending involves monitoring of the extent of arthritis in transgenic mice using a score sheet with increasing point values assigned every bit signs of pain increased in severity. Several animals were observed to prove signs of pain earlier the time when their point totals indicated they should be removed from the study. The problem was resolved by observing that when each private point value on the sheet 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 also be vigilance for unforeseen problems that may not fit the parameters designated for evaluation.

IACUC Oversight of Welfare Issues

The IACUC is responsible for reviewing and approving proposed brute studies. As investigators create and acquire new genetically altered animals, IACUC members are faced with reviewing proposals that can involve lines with a diverseness of issues such as premature lethality, altered bodily functions, increased tumor production, decreased illness resistance, altered susceptibility to microorganisms, and many others. In the Guide for the Intendance and Utilize of Laboratory Animals , one of the topics suggested for review of animal care and use protocols is "Criteria and process for timely intervention, removal of animals from a report, or euthanasia if painful or stressful outcomes are anticipated" ( NRC 1996 , p. x). Information technology is customary for the IACUC to ask investigators to list anticipated effects of the proposed manipulations and to describe the planned monitoring protocol for identifying problems.

As discussed above, when new lines are existence developed, it may be hard to predict outcomes accurately. Nevertheless, there is value in anticipating the possible problems even though the list may crave revision as the study develops. A vigilant surveillance system can exist helpful in identifying unpredicted events. The endpoints to be 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 expiry is seldom necessary to document the result or alter of a particular genetic change. When bug are encountered, the attending veterinary and the investigative team should work together to identify ways to prevent or alleviate them. Every bit new methods of limiting the timing and extent of expression of genetic changes are adult, investigators volition apply them to create more than precise and sophisticated models. As has been seen, these refinement methods can likewise prevent or alleviate many welfare problems.

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

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

Posted by: kellygeression1998.blogspot.com

Genetically Modified Animals: Should There Be Limits To Engineering The Animal Kingdom?

Abstract

Introduction

Types of Research

Cistron Discovery

Disease Models

Examination Arrangement Development

Cistron 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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