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Cloning of has been an area of huge interest over the past decade, and the applications of the technology are a cause for even greater interest. Since the production of the first live mammalian offspring following somatic cell nuclear transfer , there have been significant advances in the understanding of the molecular processes behind the method itself, and a number of different animals have been cloned since the famous dolly the sheep . Animals cloned since dolly includes cattle , cats , mice , goats , pigs , and camels . The process of somatic cell nuclear transfer is a multifactorial process, advances in each step of the process will contribute to simplifying and improving the efficiency of the technique . The steps in somatic cell nuclear transfer include, maturation of oocytes, enucleation of oocytes, transfer of donor material into oocyte, reprogramming and remodeling and finally culture and transfer of embryos .
The growing list of animals which have been successfully cloned cannot obscure the fact that cloning remains very inefficient compared to other reproductive technologies such as artificial insemination, typically only around 1-5% of all cloned embryos transferred into surrogate mothers manage to develop into viable offspring. It has been shown that factors such as oocyte source, enucleation methods as well as the reprogramming of genes and remodeling of structures contained within the nucleus contribute to efficiency of the process.
It has been shown that almost all cell types under appropriate conditions can be used as donors for somatic cell nuclear transfer . Y. Kato et al examined the cloning success rates of various somatic cell types from male, female, adult, newborn and fetal cows. They found that there was no significant difference in the percentages of blastocysts that developed from oocytes containing nuclei from the different donors, as well as this it was also found that those calves cloned from adult donor cells of 10 years and above showed adult characteristics such as wrinkles in the skin, this is was put down to the likelihood of increased mutations in the older donor cells .
Enucleation is a vital part of the process, if done incorrectly it can lead to failure of the procedure as a whole. The most commonly used enucleation method is the mechanical aspiration of the metaphase plate using micromanipulators, via this method 4% to 50% of the total cytoplasm volume can be eliminated . Comparative studies have been done into the efficiency of different enucleation methods for somatic cell nuclear transfer of different animals. One of which is that performed by Lee et al, they performed a comparative study of the enucleation methods of aspiration and squeezing in pig somatic cell nuclear transfer. They concluded that the aspiration method was more efficient method of oocyte enucleation of pigs. Chemical enucleation has also been investigated, it provides the advantage of being a simple method compared to other enucleation methods, however cytoplasts prepared from chemically enucleated oocytes induce poor cleavage rates and elsheikh et al showed that those enucleation using chemical methods such as treatment with etoposide do not have the ability to support embryo development compared to those enucleated using conventional mechanical methods therefore the value of this procedure is questionable. A number of other studies have been done to investigate other methods of enucleation one such method is that of XY clone laser system , this system involves using a laser to breach the zona pellucida prior to aspiration of the oocyte spindle, this method allows for less technicial expertise, further studies are underway investigating other procedures.
There have also been procedures that have employed a method of enucleation without the use of micromanipulators . It has been attempted to simplify the process of cloning one example of this would be to use less expensive equipment, therefore a method so called handmade cloning (HMC) has been developed. Lleterney Rodriguez et al have described one aspect of this method which involves the cloning of cattle without the use of micromanipulators, therefore simplifying the procedure as well as reducing the costs. This method involves release of the nucleus from zona pellucida by pronase digestion followed by enucleation by bisecting off the cones . This approach has been successfully applied to a number of different species . It enables for radical modifications to somatic cell nuclear transfer as well as drastically reducing costs and the need for a skilled workforce . As well as this HMC could eventually contribute to widespread application of cloning and also offers the unique possibility of eventual automation of somatic cell nuclear transfer .
The method of handmade cloning is not widely used, lleterney Rodriguez et al did produce overall somatic cell nuclear transfer efficiency of 52.6% this high level of efficiency does undoubtedly promote the need for further investigation into handmade cloning.
An improved understanding of the cellular and molecular events associated with transfer of a nucleus to the cytoplasm of an oocyte will allow for improved procedures of nuclear transfer and cloning . Not only does gene expression need to be reprogrammed, the structures within the nucleus also need to be remodeled.
A number of studies have been done to investigate whether manipulating the nucleus prior to nuclear transfer would result in an increased efficiency, this method involves the transfer of chromatin rather than the typical nuclear transfer . Sulllivan et al demonstrated the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation . They concluded that there did seem to be a greater survival rate in cloned calves when the method of chromatin transfer was used, as well as this the procedure used should be useful in investigating the mechanisms of nuclear reprogramming and improving in the efficiency of mammalian cloning . Rodriguez-Osorio et al explain that somatic cell chromatin transfer attempts to facilitate the reprogramming process by exposing the somatic cells prior to transfer, to a mitotic cell extract which in hand promotes the removal of nuclear factors enhancing nuclear remodeling.
Epigeneteic reprogramming occurs after the transfer of the somactic cell nucleus, a poor understanding of the underlying process of reprogramming has been attributed to the overall inefficiency of the process of somatic cell nuclear transfer. The reprogramming of the transferred somatic cell nucleus involves altering the state of the nucleus from its differentiated status into the totipotent state of the early embryo . A number of processes are associated with successful reprogramming of a somatic nucleus, these include erasure of cell memory, transcriptional slicing of the donor nucleus, and correct gene expression at all stages. Each of these steps involves a number of epigenetic alterations, the main ones to consider are changes in chromatin and and DNA methylation .
This process has been achieved in the mammals which have been successfully cloned and recently it has realized that human oocyteâ€™s have the ability to extensively reprogram adult human somatic cells . Due to the lack of human egg cells it has been suggested the use of animal oocytes as surrogates could be used for somatic cell nuclear transfer . However Chung et all concluded that bovine and rabbit oocytes do no support appropriate embryonic reprogramming of human somatic cell nuclei which is most likely do to that fact that DNA methylation/demethylation of the donor genome occurs in a species specific way and therefore call into the question the use of animal surrogate oocytes to generate patient-specific human stem cells .
Reprogramming in the nucleus following nuclear transfer often does not always occur correctly, frequently the donor nucleus fails to express embryonic genes and establish a normal pattern of chromatin modifications  and therefore leading to incomplete reprogramming. Blelloch et al investigated whether the methyaltion and differentiation state of the donor nucleus influenced the efficiency of reprogramming. They concluded that embryonic stem cells derivation following nuclear transfer was strongly influenced by both differentiation and methylation state of the donor cell, they demonstrated this by using hypomorphic allele of DNA methyltransferase-I, that global hypomethylation of a differentiated cell genome prior to somactic cell nuclear transfer improved efficiency. These results are of a great importance because the nuclear-derived embryonic stem cells have the potential to be used for therapy in disease, and therefore it is of practical significance to identify means of which to improve the efficiency of reprogramming .
However a study done by Enright et al showed that treatment with 5-aza-2â€™deoxycytidine, a DNA methyltransferase inhibitor had no effect on cloning efficiency. These studies seem to both cancel each other out, therefore emphasizing the need for further research.
Alterations in culture conditions for somatic cell nuclear transfer produced embryos have shown to effect gene expression  as well as development of the embryo. It has been suggested that supplementation of the culture conditions with factors that may aid in the process of epigenetic reprogramming may be beneficial in the development of the embryo. One such supplement is that of trichostatin A, which is a known inhibitor of histone deacetlyase. Inhibition of this enzyme allows for chromatin expansion and permits genetic transcription to take place, which is vital in a developing embryo. Iager et al showed that trichostatin A can improve at least one epigenetic mark in early cloned bovine embryo development. A number of other studies have also shown addition trichostatin A to the culture medium up until the blastocyst stage has increased the frequency of blastocyst formation .
A decade since the successful cloning of the first mammal, dolly the sheep, cloning has progressed immensely, improvements in the techniques involved in cloning are constantly being made. However a better understanding of areas such as epigenetic reprogramming are needed so that cloning could one day become as efficient as other reproductive technologies, which could lead to applications of the technology in a number of different areas such as gene therapy and even the prevention of endangered animals from becoming instinct by simply creating multiple copies of them.