Pet cloning” has been described as an attractive and promising strategy. The basic idea is to induce pluripotency in a somatic cell, and ultimately to transform the induced pluripotent cell into a cloned embryo. A clone embryo, capable of developing into an animal if implanted in the uterus of a host mother animal, would be generated from the somatic cell. However, despite the development of recent breakthroughs in generating induced pluripotent stem cells, “cloning” has yet to be realized in mammals. In order to produce cloned mammals, several problems must be resolved, including one that relates to the problem of “genome instability”, in which cells containing genetic mutations of somatic origin propagate unchecked in the embryo, and the other relates to the generation of a functional embryo from the somatic cell.
The present inventors have succeeded in developing a technique for obtaining a clone embryo without “genome instability”. They also revealed that embryos obtained by implanting cloned embryos into uterus can develop into a clone calf (see Non-Patent Document 1). The present inventors performed experiments with cloned embryos from various mammalian species and observed that, among the cloned embryos, blastocysts obtained by transferring cloned embryos to the oviduct of surrogate females developed into a clone calf in all of sheep, cattle, pigs, rabbits and mink, as well as in dogs (see Non-Patent Document 2). Thus, clone mammals are obtained by inducing pluripotency in the somatic cell of the fetus itself, i.e., in the “fetus-derived somatic cell,” and implanting the embryo generated therefrom in a surrogate mother.
Furthermore, among various mammals including rodents, the cloning efficiency in pigs and cattle is high; therefore, cloned pigs are also obtained easily. However, in sheep and goats (animals of the family Bovidae), the clone efficiency is low; such low efficiency is one of the factors that hinder the spread of the clone animal technique (see Non-Patent Document 2). Therefore, in order to put clone animals in practical use in many countries of the world, it is important to establish a technique capable of obtaining a clone fetus of animals of the family Bovidae.
Furthermore, in spite of the results obtained by the present inventors, the current cloning technique is low in efficiency. Such low efficiency is one of the main problems to be solved before the clone animal technique can be put into practical use.
Specifically, in order to produce a clone calf, a sheep embryo is collected from a uterus and transferred to an in vitro culture system, thereby producing cloned embryos, and then the cloned embryos are transferred to the oviduct of a surrogate mother, and thereby producing fetus-derived somatic cells. Subsequently, a clone fetus is obtained by implanting the fetus-derived somatic cells in the oviduct of a surrogate mother. It has been reported that, depending on the species, the efficiency of obtaining clones in this manner ranges from 1 to 70%, although it varies greatly among individuals (see Non-Patent Document 3).
It has also been reported that, as the cloned animals reach sexual maturity, various abnormalities and malformations tend to occur (see Non-Patent Document 4). The cause is believed to include the abnormalities or malformations present in the embryos of the original animals, but it is not known what actually causes these abnormalities or malformations. For example, when a cloned calf is obtained, the fetus can be a twin fetus; however, the incidence rate is about 30%, and the cause has been reported to be abortion after a short gestation period (see Non-Patent Document 5). However, the details of such abnormalities and malformations are unknown. Accordingly, it is not possible to prevent these abnormalities and malformations by any preventive measures. For this reason, a cloned animal should be monitored until it reaches sexual maturity; however, this is costly and time-consuming. Furthermore, the monitoring has to be carried out for a long period of time until sexual maturity, and it is not possible to detect the abnormalities and malformations only after the clone is grown up.
If it becomes possible to easily detect abnormalities in cloned embryos, various abnormalities and malformations, or abnormalities in the genetic information possessed by the clones, as early as possible, it will become possible to avoid or reduce the occurrence of abnormalities in cloned animals.
Specifically, for example, a gene having a function to induce abnormalities, such as a gene that causes a twin fetus, is not present in the embryo or cells in a fetus before it becomes sexually mature, but such gene is often produced by mutation from the somatic cell. It has been known that, in a cloned animal, when the fetus reaches sexual maturity, it begins to produce its own genes that causes various abnormalities. Accordingly, by examining the genes present in the early stage embryo or fetus, the abnormality-causing gene can be identified and removed beforehand, or removed upon discovery of abnormalities, and thereby enabling the production of a clone animal that has overcome those abnormalities.
It is particularly desirable to identify genes causing abnormalities in cloned animals, from the view point of the practical application of a clone animal. It is believed that, when the genome of an animal to be cloned is compared with that of an animal obtained by conventional breeding and selection, or the genome of the animal obtained by cloning and selection is compared, more preferable characteristics are selected in both cases; thus, a clone animal that has fewer abnormalities than non-clone animals can be created. Accordingly, it is very useful to identify abnormalities in cloned animals.
It has also been reported that various malformations are caused in cloned animals (see Non-Patent Document 4). These malformations have been reported to be frequently observed in a fetus of the Bovidae family, such as sheep and cattle; however, their cause has not been understood (see Non-Patent Document 6). Thus, in order to avoid malformations in a clone animal, it is also important to examine abnormalities in the genome of a clone embryo and fetus and to identify abnormalities in the genetic information possessed by the clone embryo and fetus. It is also important to remove the genes causing abnormalities before they are transmitted to the fetus; if the abnormality genes are transmitted, the fetus can develop into an animal with abnormalities, such as a malformed fetus.
As described above, abnormalities in a clone fetus have been reported to be caused by genes that are newly produced and released from a somatic cell. However, no cloning methods or techniques have been developed that can accurately determine the presence of abnormalities in a cloned embryo and fetus and, as a result, remove abnormalities. Non-Patent Document 1: Murakami N., et al., Nature, 432, 100-105, (2004) Non-Patent Document 2: Murakami N., et al., Nat. Biotechnol., 21, 575-581 (2003) Non-Patent Document 3: Ma H., et al., Nat. Biotechnol., 23, 1565-1571 (2005) Non-Patent Document 4: Zhi F., et al., Nat. Biotechnol., 20, 239-242 (2002) Non-Patent Document 5: Batten, J. Animal Biotechnology: An Advanced Treatise, The Wiley-Blackwell, 2008. Non-Patent Document 6: Prather S., et al., Anim. Genet., 34, 227-239 (2004)
