INTERNATIONAL JOURNAL OF LEPROSY^ Volume 55, Number 4 (Suppl.)

Printed In the U.S.A.

The Nude Mouse—Characteristics,Breeding and Husbandry

R. D. McDermott-Lancaster, T. Ito, K. Kohsaka, and M. J. Colston*

Since its discovery in 1966 ( 2 ), and thesubsequent recognition that this virtuallyhairless mutant mouse is congenitallyathymic (I"), the nude mouse has rapidlyassumed an important role in biomedicalresearch. The nude mouse is currently themost used mutant mouse; most of its ap-plications involve exploitation of the athy-mia to study basic immunologic mecha-nisms, or to study the growth of infectiousagents and tumors. It is of particular interestto the leprosy researcher, because of its ex-traordinary susceptibility to infection byMycobacterium leprae, and because the M.leprae-infected nude mouse represents a po-tentially most useful model system for theexperimental chemotherapy of leprosy.

Characteristics. Nude mice are particu-larly susceptible to viruses commonly found(as non-pathogens) in colonies of euthymicmice. The two most important are murinehepatitis virus (MHV) and Sendai virus,probably the most important causes ofwasting syndrome and decreased life spanamong nude mice. Infection of nude micewith MHV results in death of the mice with-in a few weeks, whereas heterozygous lit-termates remain free of apparent disease.At autopsy, characteristic necrotic lesionsare found in the livers of the nude mice.Sendai virus is a common respiratory path-ogen of mice. Euthymic mice may becomeill during an outbreak of Sendai virus in-fection, but the infection is not fatal. On theother hand, Sendai virus infection of nudemice usually results in death of the mice.

Although susceptibility to viral infectionsoften poses the major threat to the colony,

* R. D. McDermott-Lancaster, Department of Med-ical Microbiology, St. George's Hospital MedicalSchool, London SW17 ORE, U.K. T. Ito and K. Koh-saka, Department of Leprology, Research Institute forMicrobial Diseases, Osaka University, 3-1 Yamadao-ka, Suita, Osaka 565, Japan. M. J. Colston, Laboratoryfor Leprosy and Mycobacterial Research, National In-stitute for Medical Research, The Ridgeway, Mill Ilill,London NW7 IAA, U.K.

nude mice are also vulnerable to a varietyof bacterial, fungal and parasitic infections.A particularly common problem is infectionwith Staphylococcus sp., which may be in-troduced into the colony by the animal care-takers. Although these organisms are com-mensal in man and normal mice, in nudemice they cause formation of abscesses, par-ticularly around the mouth and eyes, wast-ing and death.

One might expect nude mice to exhibit ahigher frequency of spontaneous tumorsthan is encountered among normal mice;however, the situation is not clear, perhapsbecause nude mice have been observed forlong periods in only a very few studies. Somereports have suggested that nude mice ex-perience a higher than usual incidence ofcertain tumors, especially lymphoid andmammary tumors. On the other hand, thedevelopment of spontaneous tumors is notrecognized to be a major problem in colo-nies of nude mice.

A large number of articles have been pub-lished on various aspects of the immuno-logical status of nude mice, too many to bereviewed here. In general, nude mice lackfunctional T cells, although small numbersof cells carrying phenotypic T-cell markershave been detected. T-cell-mediated func-tions—e.g., rejection of organ allografts, andresistance to infection by intracellular or-ganisms—are deficient or absent, and non-T-cell-mediated, cellular-immune mecha-nisms may be altered. Levels of macrophageactivation and natural killer (NK) cell ac-tivity are elevated in nude mice, the extentof the elevation depending upon the mousestrain and the microbiological status of themice.

The nu gene, that responsible in homo-zygous mice for congenital athymia, has beenbred into mice together with other mutantgenes associated with immune deficiencies.Some of these double mutants arc: I) the"LASAT" mouse, which is both athymicand congenitally asplenic (S."). This mouse

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is more profoundly immunodeilcient thanis the nude mouse, although NK-cell activ-ity may be greater than that in either cu-thymic or athymic mice. Nakamura andYogi observed enhanced M. /eprae-infec-lion of LASAT mice ''); 2) the mouse pos-sessing both nu/tut and Xid/Xid (if female,or .Aid if male) genotypes. This mouse ischaracterized by deficiencies of both T-celland 13-cell function. Mice possessing the Xid/Xid genotype alone have been shown to re-spond to infection with .1/. leprac or M.marinitin as do genotypically normal mice(6); 3) the mouse possessing both nu/nu and"beige" genotypes. The beige mouse is char-acterized by deficiencies of both macro-phage and NK-cell function. This doublemutant has recently been bred at the Na-tional Institute for Medical Research, andits responses to intracellular infection, in-cluding that with M. leprac, are currentlybeing investigated; 4) the congenitally obese(ob/ob) mouse has a defective thermoreg-ulatory mechanism, resulting in a core tem-perature that is significantly lower than thatof normal mice. Although these mice do notdevelop a heavy infection after inoculationwith M. leprae, the double mutant—ob/ob,nu/nu—develops disseminated .1./. 'nal -i-lium disease involving internal organs ( 5 ).Because of the difficulty of obtaining andmaintaining these mice, M. leprac infectionof the double mutant ob/ob, nu/inc mousehas not been studied.

Breeding. Nude mice are available inwhich the nu gene has been bred onto aninbred or an outbred background. In gen-eral, outbred nude mice are more vigorous,live longer, and are more fertile than areinbred nude mice. On the other hand, ifgenetic uniformity together with a preciselydefined genetic background are required,then one must use nude mice in which thenu gene has been bred onto a backgroundof the desired inbred strain.

The nude female has poor fertility, anddoes not generally suckle her young ( 12 ).Therefore, the breeding of nude mice usu-ally employs "families," in which the maleis nude (nu/nu)and the female heterozygous(nu/+) for the nu gene. From such a cross,half of the offspring are expected to be nude,and the remaining half phenotypically nor-mal (i.e., hairy), but heterozygous for the nugene. Heterozygous males are generally more

fertile than homozygous nude males, butbreeding is not ordinarily carried out em-ploying heterozygous males and females.Only one quarter of the offspring will benude, on average; and the phenotypicallynormal offspring will include one normal(+1+) mouse for every two heterozygotes.Heterozygous females can be distinguishedfrom normals only by laborious test breed-ing with nude males.

Nude males may be mated with one ormore females. The most productive systemappears to he that of pairing one nude malewith one or two heterozygous females, whoare left together throughout their breedinglife. Alternatively, one nude male may bemated with three or more females (so-calledharems). However, to prevent cannibalism,the male must be removed before the littersare born; because female mice are most fer-tile immediately post-partum, this systemresults in reduced fertility. On the otherhand, the harem could include nude fe-males, all of whose offspring would be nude;the heterozygous harem-mates would sucklethe young. Finally, vigor and survival of thenude males may be improved by thymustransplantation ( 4 ).

The duration of the gestation period ofmice is 21 days; when the continuous mat-ing system is used, the young of the firstlitter are ready to be weaned just before thebirth of the next litter.

The nude offspring are smaller and weak-er than their heterozygous littermates, and,as a result, often do not compete success-fully. Production of nude mice is improvedby "culling" the heterozygotes from the lit-ter. Shortly after birth, nudes can be distin-guished from their heterozygous littermatesby the absence of vibrissae (whiskers). Be-cause disturbing the litter during the first 24hr after birth may lead to cannibalism bythe mother, it is the usual practice to removemost of the heterozygotes on the third orfourth day, keeping one or two heterozygotefemales to replace breeders when required.In any case, one should leave one hetero-zygote per litter, to stimulate the mother'slactation.

Finally, Fortmeyer advocates ( 3) weaningnude mice early—on the 15th or 16th dayof life. By this time, lactation has dimin-ished, and nude offspring, lacking hair, aremore vulnerable to dehydration than are

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normal mice. He has found that late mor-tality of the nude mice is reduced by sepa-rating them from the mother, and placingthem in a cage containing an easily reachedsupply of wet mouse-meal.

Husbandry. Because nude mice are vul-nerable to infection by organisms that arecommensal in normal mice, it is essentialto begin with clean breeding stock, to main-tain the cleanliness of the colony, and toprevent contact with other mice. MHV,present in many mouse colonies, will deci-mate a colony of nude mice. Insects, wildrodents and man may all carry microorgan-isms pathogenic for nude mice. Thus, al-though workers in Thailand have success-fully maintained large number of nude miceunder conventional conditions ("), a con-ventional mouse room is generally unsat-isfactory for the husbandry of nude mice.

Nude mice may be maintained success-fully under specific-pathogen-free (SPF)conditions. These conditions include sterilefood and bedding, autoclaved or acidifiedwater, frequent sterilization of water bottlesand cages, restricted access, use of protec-tive foot-wear and clothing, scrupulouscleanliness, sealing of the rooms against ac-cess of wild rodents and vermin, closure ofdrains, screening of personnel for pathogen-ic organisms, and routine screening of thecolony for potential pathogens, includingviruses (').

Small numbers of nude mice may bemaintained in a laminar-flow hood or rack,and larger numbers can be maintained in alaminar-flow room ('). A laminar-flow room,which is expensive to purchase and install,should be equipped with entry and exit airlocks; the entry air lock should include ashower, and workers should don cleanclothes, cap and mask after emerging fromthe shower. Sterile materials are introducedthrough a dip tank or double-ended auto-clave. Of course, the animals to be intro-duced into the laminar-flow facility shouldhave been bred and maintained under SPFconditions, which should be maintained.

Further protection of the animals is pro-vided by the use of filter boxes—cages thatare equipped with tight-fitting filter caps.These should be maintained and openedonly under SPF conditions.

The most rigorous protection ofnude miceis provided by housing in a germ-free iso-

lator, in which the animals may be main-tained under germ-free or SPF conditions.Germ-free mice must be administered vi-tamin K, or they may be deliberately in-fected with a defined bacterial flora designedto provide vitamin K. Isolators are fabri-cated from clear plastic, which may be flex-ible, in which case the isolator must be sus-pended from a metal frame; or the plasticmay be rigid, in which case no external sup-port is required. Isolators are equipped withfans, which introduce air from the outside(positive pressure), or exhaust air fromwithin the isolator (negative pressure); ineither case, both incoming and outgoing airarc conducted through high-efficiency fil-ters. Positive pressure protects the animalsfrom contamination, which results fromleakage into the isolator of unsterile air.Negative pressure protects the operator, animportant consideration in work with ani-mals infected with human pathogens, butexposes the animals to possible contami-nation. Work with animals housed in iso-lators is technically very demanding; food,bedding and every item of equipment mustbe sterilized before entry into the isolator,and must be introduced while maintainingsterility. Space within the isolator is limited;soiled bedding and other unneeded equip-ment and supplies must be stored withinthe isolator, until they can be removed un-der sterile conditions. Finally, the animalcaretaker stands outside the isolator, andwith his hands and arms encased by heavy,puncture-resistant gloves, a situation mark-edly limiting manual dexterity.

However the nude mice are housed, cer-tain environmental requirements must bemet. Reference has already been made tothe vulnerability of nude mice to dehydra-tion; to minimize the loss of water throughthe hairless skin, the animals must be main-tained in an atmosphere of about 60% hu-midity. Also because of its lack of hair, thenude mouse readily loses body heat and mustbe maintained at a temperature of 26-28°C.Especially for breeding, a uniform dark-lightcycle of 10 hr of light and 14 hr of darkness,or 12 hr each of light and darkness, is rec-ommended.

REFERENCESI. EDINGER, R. and GIOVANELLA, 13. C. Current

knowledge of breeding and mass production of the

888^ International Journal of Leprosy^ 1987

nude mouse. In "The nude mouse in experimentaland clinical research". Eds. Fogh, J. and Giova-nella, 13. C. Academic Press, 1978.

2. FLANAGAN, S. P. Nude, a new hairless gene withpleiotropic effects in the mouse. Genet. Res. 8(1966) 295.

3. FORTMEYER, H. P. and HASTERT, G. Breeding andmaintenance of nu/nu mice and rnu/mu rats. In:lbymuvaplastic nude mice and rats in clinical on-cology. Eds. I3astert, G., Fortmeycr, H. P. andSchmidt-Matthiesen, H. Gustav Fischer, Stutt-gart, 1981, pp. 25-37.

4. HETHERINGTON, C. M. and I 'EGAN, M. A. Breedingnude (nu/nu) mice. Lab. Animals 9 (1975) 19.

5. LANCASTER, R. D., CoLsroN, M. J., HILSON, G. R.F. and TURNER, S. M. The effect of body temper-ature and cell-mediated immunity on the growthof Mycobacterium marinum and .Ifycohacteriumleprae in mice. J. Med. Microbiol. 14 (1981)493.

6. LEVY, L., AIZER, F., BEJAR, C., LUTSKY, I. andMOR, N. Experimental mycobacterial infectionsof CBA/N mice. Israel J. Med. Sci. 20 (1984) 598.

7. LEWIN, L. and HANSEN, G. A simple, cheap barrier

system to upgrade the health status of a conven-tional rat breeding colony. Animal Technol. 37(1986) 93.

8. NAKAMURA, K. and YOGI, Y. The hereditaryathymic asplcnic (LASAT) mouse as an experi-mental lepromatous leprosy model (continued):role of the spleen in the formation of lepromatoidlesions. Int. J. Lcpr. 50 (1982) 586.

9. NAKAMURA, K. and Yom, Y. The NSF/N nudemouse, Lasat mouse and carrageenan-treated nuderat as a new model for experimental lepromatousleprosy. Proc. XII Int. Lcpr. Cong. New Delhi,20-25 Feb. 1984. Printaid.

10. PANTELOURIS, E. NI. Absence of thymus in a mousemutant. Nature 217 (1968) 370.

11. RUNGRUANG, S., RAMASOOTA, T. and SAMPATTA-VANICH, S. Study of the use of nude mice in thecultivation of M. leprae in a normal, non-specificpathogen free room at a temperature of 30-35°Cwithout air conditioning. Lcpr. Rev. 54 (1983) 305.

12. RYGAARD, J. In "Thymus and self-immunobiol-ogy of the mouse mutant nude". F.A.D.L., Co-penhagen, 1973, p. 48.

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