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Proceedings for Pain Management and Humane Endpoints

Monitoring of Genetic Engineering Studies

Melvin B. Dennis, Jr., DVM
Department of Comparative Medicine, School of Medicine
 University of Washington, Seattle, WA 98195

Introduction

Since production of the first transgenic mouse in the early 1980's, there has been an explosion in the numbers of genetic engineering studies. The Institutional Animal Care and Use Committee (IACUC) is charged with overseeing such protocols. It is aided in the review of safety issues by guidelines published by the Department of Health and Human Services. (59 FR 1994) Institutions with animal studies involving stable introduction of recombinant DNA into the germline are required to have an institutional Biosafety Committee (IBC) and an Animal Containment Specialist. U. S. animal use regulations (Dennis 1994) and general IACUC review of genetic engineering protocols involving animals have been summarized by the author. (Dennis 1998) It is the purpose of this presentation to focus on the outcomes of genetic engineering experiments and to discuss the reasons that the IACUC must monitor protocols in an ongoing fashion.

Methodologies used in genetic engineering studies consist of insertion, deletion or alteration of a segment(s) of DNA followed by observation of the animal and/or offspring to see the effects. Procedures used are described in textbooks. (Pinkert 1994) (Silver 1995) In these studies, there has been a shift away from the traditional hypothesis driven methodology to one of discovery. A segment of interest is over expressed, knocked out, or inactivated and the offspring are observed to learn the effects of the genetic manipulation.

In traditional hypothesis-driven research, IACUC review has relied upon predictions supplied by the researchers to assess the welfare issues of proposed experiments in animals. The investigator is asked to list expected outcomes and anticipated side effects of the manipulations and to explain methods to be used to prevent or alleviate pain, distress, and suffering. However, this method is inadequate for predicting the outcomes of most genetic engineering experiments. A review of the literature reveals an abundance of cases in which unanticipated and unpredictable outcomes occurred. The following cases are examples.

Case 1

In a study involving the pronucleus injection of human growth hormone (hGH) into mice, one would predict that the offspring over expressing hGH would be larger and heavier than their parents. This did, indeed, occur in the progeny. However, what was not predicted was that some offspring would also have increased liver failure, kidney dysfunction, tumor development, infant and juvenile mortality. In addition, they had shortened lifespan, reduced fertility, and structural changes in the heart and spleen. (Wolf 1995)

This case illustrates the futility of asking the investigator to predict the effects of the experiment on animal welfare. Incorporation of variable numbers of strands of heterologous DNA into a variable number of insertion sites can produce myriad outcomes, which are unpredictable. In order for the IACUC to obtain the data to make an informed decision regarding the humane aspects of such studies, there must be ongoing monitoring of the progeny. This study also illustrates that, since the numbers and sites of incorporated genes is variable, there can be countless number of experiments without "unnecessary duplication." The next experiment may produce a new phenotype by incorporating the transgene(s) into a new location.

Case 2

Mice were transfected with a drosophila heat shock gene (hsp70) and a herpesvirus thymidine kinase gene. F1 heterozygotes appeared phenotypically normal, but F2 homozygotes had loss of hind limbs, malformed forelimbs, facial clefts, and olfactory lobe defects. (McNeish 1988) This case points out the need for ongoing monitoring of suceeding generations of genically altered progeny.

Case 3

In a gene therapy study in nonhuman primates, a murine leukemia virus vector was used. It was believed that the mouse virus was non pathogenic for monkeys. Three of eight monkeys developed lymphoma within the subsequent two years. (Science 1992) This case illustrates the necessity to monitor animals following gene therapy, even when the vector is thought to be non pathogenic or replication deficient. Following genetic therapy, the host may have altered susceptibilities, or the host may harbor "helper genes" that could restore the ability of the virus vector to replicate. There should be biosafety level 2 containment of animals following gene therapy, until it is certain that the virus vector is, indeed, not capable of transmission.

Case 4

The "double muscling" gene was used to produce a transgenic bovine with faster growth rates and superior carcass characteristics.(Newman 1994) The progeny were characterized by signs of extreme fear. This case points out the need to evaluate behavioral as well as physical outcomes.

Case 5

A group had been making transgenic mice using an lck-IL-4 gene construct for a couple of years without having any problems. Then, it was observed that a new line (1315) became progressively humpbacked starting at between 3 and 6 months of age. The animals had decreased bone mass and kyphosis caused by a profound decrease in osteoblast activity. The line is now proposed as a good model for studying the previously unrecognized role of interleukin 4 in osteoporosis.

This model could have resulted from incorporation of the gene construct in different numbers or incorporation into different sites than had previously occurred. It was an unanticipated outcome that produced a useful model. It illustrates that even with the experience of several lines using a particular gene construct, the phenotype of subsequent lines cannot always be predicted.

Case 6

Transgenic sheep which can produce alpha-1- antitrypsin (AAT) in their milk at a rate of 15 gm/liter were produced. AAT is used to treat patients suffering from emphysema. The previous supply was harvested from human plasma and quantities were limited and not available for all who need it. AAT from sheep milk is cheaper and safer than from human plasma. The sheep do not appear to have welfare problems and illustrate the potential benefit of transgenic animals.

Oversight of Protocols

The cases presented above illustrate the inadequacy of confining IACUC review to the initial approval process prior to the start of a study. Since outcomes are often unpredictable, the IACUC must require surveillance or monitoring of ongoing studies in order to ensure adequate review of welfare considerations. At the University of Washington we began by using a morbidity and mortality surveillance program. It resulted in identification of transgenic and knockout lines with many unexpected outcomes, including increased tumor incidence, dopamine deficiency, diabetes, allergic encephalomyelitis, hydrocephalus, epilepsy, osteoporosis, anasarca, malocclusion, arterial wall calcification and many others.

The high frequency of occurrence of unexpected adverse outcomes has led our IACUC to draft a phenotyping protocol that could be required for investigators to obtain approval for continued breeding of each genetically altered line. Others have proposed such phenotyping protocols. (Van der Meer 1995) (Costa 1996) Under the proposed system, investigators would obtain prior approval from the IACUC for their methodology in producing genetically altered lines. However, approval to continue breeding a particular line beyond a minimal number required for maintenance and basic phenotyping would be contingent upon obtaining additional IACUC approval. The results of phenotyping would be submitted to the IACUC which would grant or withhold approval for the continued breeding of the line. The IACUC could at that time also impose conditions for continued breeding of the line. This might include treatment for conditions that compromise the welfare of the animals. Table 1 is a list of parameters proposed in the current draft. It is a suggested list and investigators could submit additional data which might be relevant to the review (i.e. immune competency or disease model data). It will be the IACUC's task at that point to weigh animal welfare considerations and potential utility of the line and decide whether to allow continued breeding of the line. In some cases, embryo or embryonic stem cell cryopreservation may be a useful alternative to continued breeding of animals with compromised welfare.

Summary

The random incorporation of injected DNA, differing helper genes, and different genetic backgrounds produces a spectrum of phenotypic outcomes, rather than a single, predictable outcome. It is impossible at the present time to predict all of the different outcomes. Therefore, an IACUC must monitor the outcomes and phenotyping data to address animal welfare considerations of these types of experiments.

References

  • 59 FR 34496 (1994). Guidelines for research involving Recombinant DNA. Federal Register, vol.59. no.34496. June.
  • Costa, P. (1996). Neuro-behavioural tests in welfare assessment of transgenic animals. In Sixth FELASA Symposium: Harmonization of laboratory animal husbandry. (ed. OÕDonoghue PN,) Royal Society of Medicine Press, London. pp 51-53.
  • Dennis, M.B., Jr, and G.L. Van Hoosier Jr. (1994). North American legislation and regulation of the use of live animals in research. In Selection and handling of animals in biomedical research, Vol.1 of Handbook of Laboratory Animal Science. (eds. Svendsen P and Hau J,) CRC Press, pp 23-36.
  • Dennis, M.B., Jr. (1998). IACUC Review of genetic engineering protocols. In Genetic Engineering and Animal Welfare: Preparing for the 21st Century. (Ed. Whitney RA) SCAW.
  • McNeish, J.D., Scott, W.J., and S.S. Potter (1988). Legless, a novel mutation found in PHT1-1 transgenic mice. Science 241: 837-839.
  • Newman, S. (1994). Quantitative and molecular-genetic effects on animal well-being: Adaptive mechanisms. J. Anim. Sci. 72: 1641-1653.
  • Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.E., Rosenfeld, M.G., Birnberg, N.C., and R.M. Evans (1982). Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300: 611-615.
  • Pinkert, C.A. (ed.) (1994). Transgenic Animal Technology: A Laboratory Handbook. Academic Press, Orlando. Sandoe, P., Forsman, B., and A.K. Hansen (1995). Transgenic Animals: The need for ethical dialogue. In Welfare Aspects of Transgenic Animals. (eds. van Zutphen LFM and van der Meer M.) Springer-Verlag, Berlin.
  • Science. (1992) Monkey tests spark safety review. 257: 1854.
  • Silver, L.M. (1995). Mouse Genetics: Concepts and Applications. Oxford University Press, New York.
  • Van Der Meer, M. and L.F.M. van Zutphen (1995). Use of transgenic animals and welfare considerations. In Welfare Aspects of Transgenic Animals. (eds van Zutphen L.F.M. and van der Meer M.) Springer-Verlag, Berlin.
  • Wolf, E., and R. Wanke (1995). Growth hormone overproduction in transgenic mice: Phenotypic alterations and deduced animal models. In Welfare Aspects of Transgenic Animals. (eds. van Zutphen, L.F.M. and van der Meer, M.) Springer-Verlag, Berlin.

Table 1: Draft phenotyping protocol.

The following will be evaluated and data submitted to the IACUC for evaluation of animal welfare for each line for which continued breeding is requested.

1. Morbidity and mortality
2. Fertility
Litter size at birth and weaningFetal Death
3. Development
Birth weight Growth rate Hair growth Development of neonatal reflexesIncisor eruption
Eyes & ears open
Stand and walk
4. Clinical parameters
Physical exam for malformationsCoat condition Nasal or ocular dischargeHemogram
Serum chemistry profiles
Tumor development
5. Simple Behavioral Parameters
Posture, climbing, & locomotionEating & drinking Grooming,Activity level, explorationAlertnessAggressionTwitches, tremors
Stereotypic behaviors
Righting
Auditory startle
Seizures
Reflexes
6. Necropsy
GrossHistology
7. Specialized testing
T & B cell functionCytokine profile Pathogen susceptibilityComplex behavioral testing
Learning testing

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