Reproductive cloning is defined as the production of a group of individuals with the same genotype by asexual reproduction, a natural occurrence in numerous plants and animals ⁽¹⁾. Over the past several years mammals have been successfully cloned by the splitting of an early embryo or Nuclear Transfer (NT) of adult somatic cells into oocytes. It is difficult to pinpoint the origin of cloning as this technology has been used by numerous scientists. In 1894, Dreisch successfully cloned a sea urchin and several years later Hans Speman successfully cloned a salamander. Briggs and King used the NT technique to clone frogs. In 1985, Steward isolated cells from the roots of a mature carrot, which resulted in the creation of a full clone. Steward also proved that it was possible to generate clones by using somatic cell NT ⁽²⁾. Cloning experiments have also been undertaken by other scientists such as Gurdon (Xenopus laevis) ⁽³⁾ and Willadsen (sheep) ⁽⁴⁾.

Nuclear transfer techniques for the cloning of domestic animals have undergone rapid development. On July 5, 1996, Wilmut et al used Roslin mehod to clone the first mammal, a sheep named Dolly. In their method, somatic cells were permitted to grow and divide. They were deprived of nutrients and induced into a suspended or dormant stage. An oocyte with removed nucleus was placed in adjacency to a somatic cell and then both cells were shocked by an electrical pulse. Both cells were fused and the oocyte was permitted to grow into an embryo. The embryo is then transferred into a surrogate mother. The birth of Dolly was considered a turning point in the history of cloning in that it showed the feasibility of producing live offspring by somatic cell NT ⁽⁵⁾.

In 1998, Wakayama et al used Honolulu method to inject enucleated oocytes with cumulus cell nuclei and produced the first cloned mouse, Cumulina, which survived ⁽⁶⁾. They removed the nucleus from a somatic cell and injected it into an oocyte with removed nucleus. The oocyte was bathed in a chemical solution and cultured. The growing embryo was then transferred into a surrogate mother and permitted to develop. Following this success, other animals such as cattle ⁽⁷⁾, goats ⁽⁸⁾, dogs ⁽⁹⁾, domestic cats ⁽¹⁰⁾, rabbits ⁽¹¹⁾, horses ⁽¹²⁾, rats ⁽¹³⁾, ferrets ⁽¹⁴⁾ and zebra fish ⁽¹⁵⁾ have been successfully cloned.

In Iran, cloning experiments were performed by Kazemi Ashtiani and colleagues at Royan Institute. They produced the first cloned sheep in Iran and the Middle East ⁽¹⁶⁾.

Wakayama et al developed a mouse somatic cell NT method by injecting donor nuclei into enucleated oocytes using a piezo unit ⁽⁶⁾. For other researchers, obtaining results with piezo unit was difficult. Currently, numerous laboratories have reported the continual reproduction of cloned mice, including cumulus cells ⁽⁶⁾, tail-tip cells ⁽¹⁷⁾, Sertoli cells ⁽¹⁸⁾, fetal cells ^{(19, 20)} and chimeric mice ⁽²¹⁾.

Cloning technology has numerous applications in industry, agriculture and medicine. This technology is considered as a research tool in basic biology ⁽²²⁾ and is a tremendous tool for the study of processes such as nuclear reprogramming, imprinting genes and gene activation ⁽²³⁾. The adaptability of the mouse has been introduced as an experimental model of choice, which can be used in the development of new applications ⁽²⁴⁾.

Currently, progress in biological research is dependent on the capability of researchers to skillfully perform techniques in the field of stem cell and NT. In the present study, we described the successful cloning of a mouse by using piezo-assisted NT.

In this study, we reported the production of live mouse pups by a stable NT method in which donor nuclei from cumulus cells of B6D2F1 mice were directly injected into enucleated oocytes using a piezo-actuated micromanipulator. The resultant clones derived from cumulus-cell nuclei were not contaminated, as the transferred embryos were not the result of in vitro fertilization. Pseudopregnant mice (NMRI, albino) were allowed to mate with vasectomized males (NMRI, albino) which were confirmed to be infertile. In the rare event of fertilization by a vasectomized male, the newborns would be albino. We transferred two-cell embryos into the oviducts of foster mothers. The surviving four mice had black coat colors. Where possible, assumed clones were sexed and determined to be female. Additionally, we tested all culture media and reagents, and determined their correctness.

Pioneers in the field of mouse cloning have stated that microinjections using the piezo unit, as a new technique, are of greater benefit in comparison with other micromanipulation techniques, such as ES cell injection into blastocysts or pronuclear DNA injection ⁽²⁵⁾. However, personnel will need several months to achieve an adequate level of skill in the required techniques. Much practice is necessary to produce cloned mice; otherwise it is impossible ⁽²⁶⁾. According to reports, when a highly skilled person performs mouse cloning experiments using cumulus cell nuclei, 99-100% of the oocytes will be enucleated successfully and 80-90% will survive nuclear injection. Of these, 70-80% should survive activation, 60-70% show pseudo-pronuclear formation and 50-60% will cleave to the two-cell stage. The rate for the production of full-term offspring ranges from 0 to 10% ⁽²⁵⁾.

In the current study, with adequate technical expertise of more than one year, we could get the acceptable results in all stages of our experiment. The rate of two-cell stage development for reconstructed oocytes in the current study was higher than previous studies that used different culture media such as CZB ^{(6, 25)} and KSOM ^{(25, 27)}. Therefore, we have shown that mWM is an appropriate medium to support the development of cloned mouse embryos.

In the current study, the donor cells were cumulus cells. There was one foster mother with five fetuses in our study. In this regard, our result is similar to the result obtained by Satoshi et al ⁽²⁷⁾. They have reported that following oocyte activation, treatment of oocytes with 5-50 nM TSA increased in vitro development of somatic cloned embryos from 2- to 5-fold, which was dependent on the donor cells, including cumulus cells. The success rate of mouse cloning from cumulus cells was increased by over 5-fold without any obvious abnormalities. In their study, treatment with 5 or 50 nM TSA led to multiple conceptions. In one case, five fetuses of one foster mother were born full term. However in this study, the percent of live offspring was approximately 1%. In the study by Satoshi et al ⁽²⁷⁾, it was 6%. Most likely, additional experience is necessary in order to improve the cloning experiment results in our laboratory. Since accuracy of our experiment was detected and confirmed by comparison of sex and coat color of cloned mice with source of donor cells, we didn't perform any genetic test for more evaluation.

The adaptability of mice as a small mammal has resulted in its use as an appropriate experimental model for the study of new cloning applications and strategies ⁽²⁴⁾. The information available on their reproduction, development and genetics is unmatchable to other laboratory animals ^{(28, 29)}. At present, numerous advanced techniques have been established by the use of mouse embryos. The mouse has a short generation period, which could be advantageous for the examining of long-term genetic effects that occur during biological manipulation, such as cloning ^{(30, 31)}.

Our study resulted in the production of cloned mice by somatic cell NT using piezo-actuated micromanipulator. This accomplishment is another important event in the technology of cloning in Iran.