Mice as Basic Research Models

Mice are barnyard animals too, albeit not in the conventional meaning of the phrase. The mice discussed here call the laboratory home, rather than the farm, and their inclusion in this chapter is meant to highlight the underlying worth of basic or pure scientific research on GM animals. Laboratory mice (Mus musculus) have been at the forefront of the GM revolution, consistently yielding new insights on mammalian reproductive technologies and cellular operations.

Mice are favored for experimentation because of their small size, short generation time, large litter size, ease of husbandry, and the longstanding commercial availability of purebred strains for genetic analyses. In 2002, the complete sequence of the mouse genome was published, and this knowledge will further assist researchers who use mice as experimental models in genetic engineering. This essay describes key historical scientific breakthroughs on mice that will provide a backdrop for subsequent essays on GM methods and whole-animal cloning in livestock and other bona fide farm animals.

In principle, one way to engineer a transgenic mammal is to isolate a one-celled embryo (zygote) from a newly pregnant mother, fuse to it a desired piece of foreign DNA, put it back into the reproductive tract of an adult female, and hope for the best. In 1980, Jon Gordon and collaborators did precisely that. They microinjected short pieces of viral and bacterial DNA into one-celled mouse embryos, implanted the embryos into surrogate mothers, and later witnessed the birth of transgenic baby mice. Because the genetic transformations were initiated at the single-cell stage, after which the mitotic cell divisions occurred during embryonic development, each resulting mouse carried the transgene in all of its cells, germline as well as somatic. Thus, during normal sexual reproduction, the GM mice could transmit the transgene to their own natural offspring. Similar genetic engineering experiments with mice soon showed, also for the first time, that transgenes inserted by this method could alter important phenotypic features of GM mammals.

However, mammalian zygotes are tiny (each about one-tenth of a millimeter across), hidden deep inside the female, relatively few in number (compared to those of many fish, for example), and difficult to reimplant and coax to full term through a pregnancy. So, although this direct approach to genetic modification is conceptually straightforward, it can be difficult in practice. Accordingly, researchers also began to explore alternative GM routes, the two most successful of which proved to involve embryonic stem (ES) cells and cloning via nuclear transfer (NT).

Since 1885, it has been known that certain types of animal cells can grow and proliferate in artificial culture, such as a Petri dish, under suitable nutrient conditions. A century later, some special properties in this regard were discovered for ES cells in mice. Embryonic stem cells are generalized (undifferentiated) cells, taken from an embryo, that retain pluripotency—unbridled capacities to specialize later into new muscle cells, liver cells, heart cells, germline cells (eggs or sperm), and so on. In the early 1980s, researchers discovered that mouse ES cells properly cultivated in a Petri dish can be coaxed to retain this natural flexibility for a number of cell divisions. The pluripotency of ES cells, plus their relative ease of artificial culture, made these cells obvious targets for genetic engineering. The idea was to transform large numbers of cultured ES cells, for example by soaking them in foreign DNA, which the cells occasionally take up. Transgenic cells then would be returned to a multicellular natural embryo in a pregnant female. Researchers began accomplishing all of these feats with mice in the early 1980s.

Most of the resulting GM mice were chimeras; that is, they showed mixtures of two different genetic cell types: transgenic cells tracing back to the GM cell cultures and non-transgenic cells from the natural embryos. In effect, the live animals were only part GM. This also meant that the engineered mice transmitted their new genetic endowments to subsequent generations only when the transgenes happened to end up in cells destined to form an animal's eggs or sperm. This was not much of a practical problem in laboratory mice, which could be bred quickly, cheaply, and in abundance. But it could create difficulties for perpetuating GM dynasties in large, long-generation creatures like cattle.

For livestock engineering, this ES-cell method was less than ideal for another reason, too: Despite intense research, no one in the 1980s had succeeded in artificially culturing pluripotent or totipotent ES cells (those capable of directing the development of a full-term organism) from any mammal other than mice. The problems later were circumvented to some extent, but failures at the time prompted efforts to engineer strains of GM farm animals by yet another route, by cloning via nuclear transfer. Mice again paved the way.

In a 1981 paper, Illmensee and Hoppe announced the birth of three cloned mice by NT. In each case, the researchers extracted the nucleus from a toti-potent embryonic cell by micropipette, transplanted it into an enucleated (nucleus removed) egg cell, allowed the newly generated cell to divide for several days in artificial culture, and then returned the early embryo (blastocyst) to the uterus of an adult female who later gave birth to a baby identical in genetic makeup to the original donor cell. At least, that's what the authors claimed. Despite repeated attempts over the next decade, other scientists were unable to replicate these experiments, and to this day nobody is quite sure why.

But in a historical sense, it hardly matters. The amazing claims in the 1981 paper spurred research efforts that led, two decades later, to the verified production not only of cloned mice by similar NT methods, but also to the routine production of cloned GM livestock (see following essays). Nowadays, elements of both the ES and the NT approaches often are used jointly when engineering GM farm animals. Embryonic stem cells are isolated from natural embryos, grown in artificial culture, and genetically transformed in a Petri dish. Nuclei from these transgenic cells are injected into enucleated eggs which then are reimplanted into the womb of a female. Later, if all goes well, a baby is born whose nuclear genome is a clone of the original transgenic cell.

As so often proves true, basic genetic research, in this case on mice, had unforeseen practical applications. As a way of perpetuating transgenic (or non-

transgenic) barnyard animals with desirable genetic characteristics, whole-genome cloning, alone or in conjunction with other GM procedures, promises to alter the fundamental hereditary ground rules of farm-animal husbandry. The use of mice as model systems for genetic experimentation must be considered an unqualified success in generating important scientific insights for biotechnology.

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