Perspective of zona-free method use in farm animal cloning

The review is dedicated to one of the relevant and widely discussed topics of modern biotechnology, namely cloning of mammals. Particularly, the success and problems of the somatic nuclear transfer (SCNT) are discussed herein. The advantages and disadvantages of a commonly used SCNT and zona-free modification are compared based on special publications and the data obtained in our experiments. The most promising targets for the SCNT are reproductive cloning, therapeutic cloning and fundamental science. Conservation of rare and endangered species is also in focus. Nevertheless, to date the cloning application is still relatively limited. One of the reasons is a low yield of healthy offspring in mammals, for example, average yield in cattle is about 9 % of cloned embryo transfers result in birth of healthy offspring. It is assumed that deviations in the development of cloned fetuses are caused by disorders in genomic reprogramming of a somatic cell nucleus, which results in significant disturbance of gene expression particularly in placenta. Even though there are several practical techniques that allow to increase effectiveness of SCNT, reprogramming of the nucleus demands further study as one of the fundamental problems of developmental biology. Second problem that hinders practical application of SCNT method is complexity of the conventional technique, which was introduced about 30 years ago by S.M. Willadsen (1986). Since then the technique has been applied almost without any variations. At the same time significant progress has been achieved in the so called zona-free nuclear thansfer method (zona-free NT), where oocytes are freed from zona pellucida before enucleation. This method was successfully applied for the first time by T.T. Peura et al. (1998). They used blastomeres of bovine embryo for electrofusion. The method was also effective for creation of cloned embryos of pig (P.J. Booth, 2001), sheep (T.T. Peura, 2003), cow (P.J. Booth et al., 2001), horse (C. Galli et al., 2003) using somatic cells. We have improved zona-free NT method for cattle embryo cloning (G.P. Malenko et al., 2006). In the available publications there are no references about zona-free NT use by other researches in Russia while worldwide it is considered more simple, effective and reproducible method compared to a conventional one (I. Lagutina с соавт., 2007; B. Oback с соавт., 2007). Preparation of cytoplasts by enucleation of zona-free oocytes can be carried out without fluorescent dyes with effectivness of 95-100 % and preservation of 96-97 % of ooplasm volume (M.I. Procofiev et al., 2007). Electrofusion rate of zona-free cytoplasts and somatic cells is 95-100 % (I. Lagutina et al., 2007; G.P. Malenko et al., 2007) compared to 60-70 % achieved during conventional cloning (I. Lagutina et al., 2007). Blastocyst yield is equal or higher then yield produced by the conventional method. Embryo transfer results are comparable for both methods. Zona-free NT method may further increase the output of cloned embryos and offspring of the farm animals due to the simplicity and high effectiveness. Since the unique gene combinations of elite bulls’ genotypes can not been copied by natural reproduction, their cloning is promising, particularly by means of zona-free NT-SCNT. SCNT application also seems to be the most prospective in animal transgenesis.