The Laboratory Opossum (Monodelphis domestica) in Laboratory Research

John L. VandeBerg, Ph.D., is Scientific Director and Edward S. Robinson, Ph.D., is a scientist at the Southwest Foundation for Biomedical Research, San Antonio, Texas. John L. VandeBerg, Ph.D., is Scientific Director and Edward S. Robinson, Ph.D., is a scientist at the Southwest Foundation for Biomedical Research, San Antonio, Texas. The South American gray short-tailed opossum, Monodelphis domestica, is a small (80-120 g), nocturnal marsupial native to Brazil and adjacent countries. It is docile, breeds readily in captivity, and produces large litters (typically 6-13). Since the first M. domestica were imported into the USA in 1978, efforts to develop this species as a laboratory marsupial have been highly successful ( VandeBerg 1983 , 1990 ). The standard laboratory diet is a commercial pelleted fox food provided ad libitum, and the animals are maintained in polycarbonate or polypropylene rodent cages under unremarkable conditions ( VandeBerg 1990 ). This is the only marsupial species that has been produced in captivity in very large numbers, i.e., tens of thousands. Breeding colonies currently exist at academic and research institutions in Germany, the United Kingdom, Brazil, Australia, and at several locations in the USA. Because of its many advantageous characteristics as a laboratory animal and its economical production in captivity, M. domestica has become a prototype species for basic research on marsupial biology, in much the same way that the laboratory mouse is a prototype species for basic research on the biology of eutherian or placental mammals. Consequently, we now refer to M. domestica as the laboratory opossum. The status of M. domestica as a laboratory animal was reviewed by VandeBerg (1990). Much progress has been made since then, both in basic biological research with this species and in its development as an animal model for research on human diseases. The present review focuses on recent progress. Table 1 summarizes the origin of the current population of laboratory opossums. There were seven separate introductions of animals into captive breeding between 1978 and 1993; the animals came from four different localities in three states of Brazil, and a single locality in Bolivia. In total, 38 animals were imported, of which 28 have contributed to the current gene pool as founders. Only portions of the genomes of each of the 28 founders are still present, because some genes are lost in each generation by chance. For example, between 1978 and 1984, a computer simulation based on the pedigree structure of the colony estimated that 31% of the genetic material of the original nine animals imported in 1978 already had been lost ( MacCluer and others 1986 ). Therefore, the genetic variability present in the colony is much less than that which was present in the 28 founders. van Oorschot and others (1992a) have described in detail the geographic origins of the founders obtained by 1988 and genetic differences between the groups descended from them. Founders of the laboratory population of M. domestica All localities are in Brazil, except Santa Cruz, which is in Bolivia. Exu is 45 km southwest of the city of Crato. Piraua is 25 km west of the city of Timbafiba. Conselheiro Mota is 37 km west of the city of Diamantina. Bracha 3 is 27 km southeast of the city of Santa Cruz. Figure 1 illustrates the locations at which the founders were trapped. Some of these populations are separated by major rivers and mountain ranges which may substantially inhibit the transfer of genetic material between populations. Map of South America showing the sites at which each of the 5 sets of founders were trapped. The numbers 1 through 5 refer to the laboratory codes for the population sampled at each of the 5 sites (see Table 1 ). Approximate distances between adjacent sites are indicated in kilometers. Founding animals from the five populations are designated in our laboratory as from Populations 1, 2, 3, 4, or 5, respectively, as are any animals descended entirely from founders of any one population. Population 1 is designated as the reference population, and interbreeding among populations has been restricted to Population 1 and any one other population. Animals descended from ancestors from Population 1 and Population 2, and having less than 30% of their genetic material derived from Population 1, are designated as members of Admixed Population 2. Admixed Population 3 has up to 37.5% of its genetic material from Population 1, and Admixed Population 4 has 50% of its genetic material from Population 4. The Bolivian animals (Population 5) have not been crossed with the Brazilian stocks. More than 29,000 progeny have been weaned in our colony since 1980. Currently, the colony has approximately 2,400 individuals of which many are used in experimental research rather than in the breeding program. The breeding colony consists of about 700 adult females and 450 adult males, and it produces approximately 4,600 weanlings per year, in addition to sucklings used experimentally before weaning. Approximately 300 individuals are provided to other institutions each year as experimental subjects or breeding nuclei. The rest are used as research subjects, or as replacement breeders to perpetuate stocks or to continue the development of inbred strains. Detailed records have been kept on each animal in the colony, which is fully pedigreed, and the data base is computerized. Some pedigrees extend back as many as 22 generations. Because of the low number of founders and the large number of animals produced over many generations, a limited amount of inbreeding has been unavoidable in the colony at large. In addition, some groups have been purposely inbred over several generations to develop partially inbred strains. Brother-sister matings were used in initial attempts to develop inbred strains. One stock achieved an inbreeding coefficient of 0.911 before it was lost, but attempts to produce fully inbred strains (F = 0.986, equivalent to that achieved after 20 consecutive generations of full-sib matings) have not succeeded because of rapid decline in fecundity and infant survival after five or six generations. We hypothesize that the low recombination rate in females by comparison with eutherian mammals ( van Oorschot and others 1992b , 1993 ) inhibits the formation of new gene combinations sufficiently quickly in brother-sister mating schemes to enable the acquisition of gene combinations compatible with an inbred state. However, we are succeeding in gradually increasing the inbreeding coefficients in partially inbred stocks by less severe inbreeding schemes, and are hopeful of achieving fully inbred strains in this manner. At the present time, the colony has 34 partially inbred stocks. Of those that continue to have high fecundity and infant survival rates, the most highly inbred has an average inbreeding coefficient of 0.674. Approximately 600 members of the colony have inbreeding coefficients in excess of 0.6. Since more than 60% of their gene loci have alleles that are identical by descent, these animals are valuable for research designed to detect recessive genes for susceptibility to experimentally induced diseases. They also are especially useful for intercrossing and backcrossing for linkage analysis and gene mapping purposes. Animals superfluous to our own research needs have been available for distribution to other laboratories since 1984, and the species has already made important contributions in many areas of basic and biomedical research. The annual number of publications, which were indexed in BIOSIS Previews (all life science subjects), about M. domestica rose from 0 in 1978 to 14 in 1986 ( VandeBerg 1990 ). The number has grown steadily since then by approximately five per year: 45 were indexed in 1992 and 54 in 1993, making a total of 276 indexed publications since 1978. The possibility that this increasing rate of expansion of the scientific literature on M. domestica is simply a reflection of increasing rate of publication on all marsupial species is not substantiated by a count of the numbers of publications concerning Macropus eugenii and Sminthopsis crassicaudata, two other commonly used laboratory marsupials; for both of those species the annual number of publications has remained approximately constant with means of 25.2 and 4.2, respectively, during the 5-year period from 1989 to 1993. Some areas of investigation have developed because of the marsupial characteristics of this new laboratory animal; for instance, birth after a short gestation enables manipulation of young at an immature stage of development ( Tyndale-Biscoe and Janssens 1988 ; Janssens and others 1995, Saunders and Hinds, in press, chapter 4). Others arose because of the value of an out-group of mammals for comparative research strategies; for example, marsupials exhibit paternal X-chromosome inactivation rather than random X-chromosome inactivation, as is characteristic of eutherian mammals. Laboratory opossums are used as a model for comparative research on these divergent strategies to accomplish balance between sexes in expression of X-linked genes (reviewed by VandeBerg and others 1987 ). Still other areas have arisen simply because preliminary studies on physiological systems of M. domestica revealed research opportunities that did not exist for the conventional laboratory animal species that were available. These other research opportunities are not related to the marsupial characteristics of laboratory opossums, but arose simply as a consequence of the existence of a new laboratory animal with its own species-specific characteristics. Ultraviolet radiation induced skin and eye cancers and dietary induced hypercholesterolemia, discussed in this review, are diseases for which M. domestica has become an important model species for reasons unrelated to its marsupial characteristics. The incidence of human melanomas is increasing at an alarming rate, indeed at a faster rate than any other type of cancer except lung cancer in women. Ultraviolet radiation (UVR) is believed to be a major causative factor. Unfortunately, research on the role of UVR in inducing melanoma in humans has been hampered by the lack of a mammalian model in which melanoma can be induced by UVR alone. UVR is not a factor in the induction or promotion of melanoma in the Sinclair swine model ( Greene and others 1994 ) and in mice, UVR must be combined with chemical carcinogens to induce melanomas ( Kripke 1979 ). Although a newly developed transgenic mouse appears to be a promising model for research on UVR-induced melanomas ( Mintz and Silvers 1993 ; Klein-Szanto and others 1994 ), the laboratory opossum has the distinction of being the only naturally existing mammal, other than humans, known to be susceptible to melanoma in response to UVR alone. In humans, some types of melanocytic nevi or moles are a risk factor for melanoma; occasionally a cell in these nevi can undergo transformation and become a melanoma cell. Conditions for the production of melanocytic nevi by repeated of adult laboratory opossums and to UVR were developed by and (see and others , and The in our laboratory to a of at a of per This is equivalent to at from the for 1 to on the of the animals after the initial but severe In our the incidence of melanocytic nevi after three per was after and after 45 ( VandeBerg and others ). the same was in weanlings rather than young the incidence was only after 45 ( and others ). cell were developed from melanocytic nevi induced in the with ( and others 1993 ). revealed that three of of 3, 5, This that of these be an in the induction of melanocytic In an to the and to produce animals with nevi as of we a for laboratory opossums between birth and of to become ( and others 1994 ). This on the that M. domestica are the are fully on the of their The the UVR a up at the of a on the a of many individuals can be in a short time, the for or Although the to be the combined of all have an incidence of of animals or up 1 after ( and others 1994 ). are currently being and are in progress to susceptibility to formation is under genetic In some we have animals that had been as The and the weanlings three per as in the Of the animals that had a at developed melanoma after up to 45 of In each the original to in and to one or more derived from from an provided of in this model ( and others 1994 ). only were highly and in of but were and produced after into to or UVR is believed to be a factor to of of the and to formation of by in and ). The laboratory opossum is susceptible to the development of these after repeated to low of and an model for investigation of the biological In many individuals of this and are by the development of Although UVR-induced cancers are and in humans ( and others 1986 ), those that develop in M. domestica a valuable We developed a for of in by UVR from through and to an highly stage ( VandeBerg and others ). The development of most types of is by is a in of M. domestica during transformation of into in the the of the the and become most at of The through the into adjacent and at much of the the and the to the is known about the by which UVR these or ( and have been in experimentally induced animal and naturally human and of may a role in the development of UVR-induced in M. domestica ( and others 1992 ). it appears that the opossum gene can be in a to the genes of eutherian mammals ( and others 1993 ). One of to at of the UVR and 45 ( VandeBerg and others ). Although the is small for genetic because only a single generation was of of were of was with a of to after 45 was with the to It be important to the of with a but already it is that susceptibility to UVR-induced is highly in this population. the single of and in and characteristics of are among the most important risk research on since the has our of of human Some of that progress is to the of animal mice, and of the and progress in research has related to as hypercholesterolemia, rather than the which are substantially for the of that exhibit genetic in to dietary are to the genes that are believed to to variability in this physiological to with the laboratory opossum that this species a and valuable model for this It in response to dietary and and that appears to be by a single recessive in humans and other animal can to in M. domestica and VandeBerg, our other animal model of a dietary induced that is both as as in M. domestica and as highly by a single of this gene and of its of may to a of in human characteristics. the characteristic of M. domestica may it a model than conventional laboratory animals for research on physiological of dietary and The in laboratory opossums a standard laboratory diet is 22 ( VandeBerg and ). For experimental dietary the diet is with from the of to and with from to This of dietary to about of from equivalent to the in many The of is than the in but not the high of the of human laboratory opossums have this diet for the population data for are In two the means of the low were and ( VandeBerg and ; and VandeBerg 1992 ). The means of the high groups in the two studies were and animals in the of to after this diet for VandeBerg, For the of human in the of are to be and in excess of high risk of for have of large of data from animal and human subjects that the amount of by very low and low is a causative factor in that by high is (see and others and of risk of is the of low to high In M. domestica, which are low this is constant of and for the standard and However, the is in the high the from to ( and VandeBerg 1992 ). is a characteristic of low The characteristics of M. domestica under these two dietary have been in detail ( and VandeBerg 1992 ). The to human in characteristics and that for the laboratory opossum model can be in to human The initial pedigree data were but were with the that after dietary were by a single recessive gene ( VandeBerg and ). More data from individuals have been by analysis ( and others 1994 ). This analysis on low and that in animals the diet is as a major The indicated that of the total in the population is to at this gene a high by a single gene to any is and the value of the M. domestica One or more other genes also to low because the for of the total the model may enable an of a major gene with genes and in a of has been to marsupial gene and the have been in to mammalian mapping data have been derived by in of human gene to of marsupial and some were obtained by cell genetic All data derived for marsupials from these are from and ( and others 1993 ). The laboratory opossum a to the comparative to mammalian because American and marsupials are only having as separate for an estimated ( and others ; and others ). In it is estimated that to have since the of the marsupial and eutherian ( and others 1990 ; and 1992 ). Therefore, in have been between of eutherian mammals or between marsupials and eutherian the in M. domestica can be used to which the the of biomedical the more important of gene mapping is linkage with For that we have our gene mapping strategies on linkage The is to a linkage for genes that physiological physiological to and susceptibility to experimentally induced diseases. genes can be in the M. domestica and the is sufficiently to of to the human then it may be to those genes from human and to in in to human physiological M. domestica, all other has a low number of = in this which are large and for analysis ( and others 1983 ). for high and have been and can be per of ( and others 1993 ). efforts to genetic on in from Population and Population 2. have been by and and was in of from ( and others 1989 ; van Oorschot and VandeBerg 1989 ; VandeBerg 1990 ; and others 1990 , , 1992 ; and others 1992 , van Oorschot and others , , 1993 ). The of loci for these is approximately per This of is with that in most eutherian species and than that in of loci per and others 1979 ). and three more populations 4, and 5) to be The founders of these three populations were distances from the founders of Populations 1 and ( Figure 1 ), we the of more in the laboratory population of M. domestica to in of M. domestica have been from several human used in are promising and one by a for the has been as with This two in M. domestica One consists of two alleles by and Animals of from Populations 1 and of and for the two alleles ( van Oorschot and others ). The other is a and ( and others 1994 ). All animals from Populations 1 and were for the the two founders from (Population were for the Although the data are limited to be as of the of differences in gene between the populations ( and others 1994 ). the number of available for linkage analysis more and to the of animals for the of linkage are being used to random for by the have that as that these be a useful to and random in a linkage that is for development is the of M. domestica for for this linkage groups have been in M. domestica, and both exhibit much recombination in females than in ( van Oorschot and others 1992b , 1993 ). and exhibit a recombination rate of in females and are in males, and and the exhibit a recombination rate of in females and are in and have not in and also are very in eutherian mammals ( and others ; and others 1983 ; and others ). Because of the estimated of of marsupials and eutherian these linkage groups are in to mammalian and are not in or and are in and humans and arose by gene and is with in but not in humans on 5, on ( and others 1993 ). on these it that the is to the between marsupials and eutherian mammals and was during of the human The in recombination rate is with of which revealed that are the in but more the of the in ( and others 1988 ). These recombination and characteristics those in an Sminthopsis ( and others 1986 ). The in recombination be valuable for and mapping matings with can be used in designed for We to random breeding stocks of animals to Populations 1, 2, and 5, Population 5 become inbred quickly to enable its with population. In addition, random breeding stocks of Admixed Populations 2, 3, and 4 be These populations enable the of as much genetic variability as is stocks that may different purposes. Although some of the 34 partially inbred stocks be lost, we the of fully inbred strains by gradually increasing the inbreeding coefficients of the existing stocks. The of large numbers of animals for susceptibility to UVR-induced nevi has to on a genetic analysis of this Because the is to in animals as matings based on can be the a genetic to susceptibility or the model research on the by which UVR can to become nevi and The animal model also may be useful for and for initial investigation of this type the of the model for this ( and others 1993 ). The melanoma cell are as experimental model systems for melanoma research because are derived from that were and by UVR and the to produce most human melanoma cell currently These cell be useful for the and development of conventional studies of and in There is also the of successful of cell in this for in in and for ( and others 1994 ). UVR-induced in the of M. domestica a valuable for studies on and transformation in this and In is by a balance between that new and that inhibit In are as a of production of and of ( and others 1994 ). The of the eye of M. domestica is a and readily available for in studies on the of that are known or to or inhibit For example, it be to the of known of ( and 1994 ), the and ( and others 1994 ). M. domestica also in It be to in and in strategies to the of or and in this new In addition to a model for genetic analysis of dietary the laboratory opossum also has for research on other dietary that For example, the model can be used to the of different types of dietary in with one and in with dietary the major gene and become and this as a model not only of biomedical but also of to by and by diet In addition, the model has for the of Although linkage groups have been in all marsupial species M. domestica is for linkage mapping because of its high data that Population 5 may be different from the Brazilian populations of M. domestica in gene and that the Bolivian population may be for some alleles not present in the Brazilian population We to produce and progeny from matings between Populations 1 and 5 and to this of animals as the of linkage mapping We that the Populations 1 and 5 with the of to random and the of linkage mapping in the Although the laboratory opossum on the research less than two its value to existing research in comparative biology and is the of data that now for this its and the number of it in experimental the are to new opportunities to this prototype laboratory We for to of data and of the were in by and a from the and