Somatic Cell Nuclear Transfer Process

Somatic Cell Nuclear Transfer Process Attempts at cloning a mammal can be traced back to 1979, where the scientist Steen Willadsen successfully cloned a sheep embryo using nuclear transfer [1]. Since then numerous attempts have been made to replicate these results. Notably the birth of Dolly the sheep (1996) was a major development in this field; as she was the first mammal to be cloned from a fully differentiated somatic cell, using somatic cell nuclear transfer (SCNT) [2]. This essay will describe the process of somatic cell nuclear transfer in light of mammalian cloning and the risks it poses to mammalian reproduction. The fertilization of mammalian gametes through natural reproduction is limited by the ability to preserve desirable traits after the extinction of an individual. Moreover, the reproductive success of natural fertilization is limited by the gestation length, estrus cycle, the efficiency of insemination during intercourse and Hayflick limit [3]. Furthermore, these limitations are chiefly important in livestock agriculture; where desired traits and alleles are more favourable for propagation. SCNT enables us to extract the nucleus of a fully differentiated somatic cell (diploid cells) and introduce it into an enucleated mature oocyte which is allowed to develop into an embryo; that is genetically identical to the host cell [4]. Other variations to this method are practised even though they all rely on the same principles. By this process, the limitations stated above become insignificant as specific mammals with the desired traits can be cloned to preserve the genome. However, this technique is still undeveloped and the success in producing cloned offspring is low. The success rate of SCNT is dependent on several factors; namely, selecting the right donor cell that will be most efficient to the nuclear transfer. In this process, fully differentiated somatic cells are selected based on their cell-cycle state and age. The G0 phase is most desired when selecting the donor cell as it has been shown to be the most effectual donor [5]. Conversely, deprivation of nutrient to the donor cells growing in vitro can also induce the cells to adopt the G0 resting phase. The age of donor cells also contribute to the success of cloning, the more aged the donor cell the less efficient SCNT becomes. Additionally, donor cells that are derived from more genetically diverse species are favoured, as it has been shown that cells obtained from inbred animals are less likely to be successful in cloning [6]. However, these factors are only relative to the limited species that have been examined and more factors may come to light as other species such as primates are subjected to SCNT. Once the donor somatic cells are identified, they are normally extracted from the skin of the donor mammal, using needle aspiration and avoiding unnecessary strain on the donor animal. Oocytogenesis is the process in which females produce oocytes. SCNT uses mature oocytes in metaphase-ll which are collected from the ovaries of the required animal [7]. The mature oocytes are enucleated using micromanipulation which penetrates the zona pellucida and removes the nucleus. There are two alternative routes which can be adopted when manipulating the process of the insemination of the nucleus donor cells into the mature oocytes. First, the Honolulu technique (developed by Wakayama) which uses brain cells, cumulus cells and sertoli cells as donors that are naturally in the G0/G1 phase. The nucleus of the somatic cell is aspirated and directly micro-injected into the oocyte using a piezo-impact pipette; which penetrates the zona pellucid and delivers the nucleus into the enucleated oocyte [8]. The oocytes are subsequently activated by exposing them to a medium containing Sr+2 that also contains cytochalasin-B which acts to prevent the formation polar bodies. Figure. 1[9] shows a diagrammatic representation of the Honolulu technique, highlighting that the nucleus is directly inserted into the mature oocyte. Secondly, the Roslin technique (used to create Dolly the sheep) cultures donor cells in vitro and deprives them of nutrients; forcing the cells to adopt the G0 phase. Subsequently, the enucleated oocyte is aligned next to the donor cell; such that the oocyte and donor cell are parallel to one another. Pulsating electrical currents are applied to fuse the oocyte and donor cell together, by inducing pore formation of the cell membrane [10]. Figure.1In the Honolulu and Roslin techniques the use of chemicals and electrical pulses induce the activation of the oocyte, which can subsequently develop into an embryo which is implanted into a surrogate host for progeny development. The activation of the oocyte induces major reprogramming of the differentiated donor nuclei back to its totipotent state [11]. This process is extremely intricate and the full biochemical mechanisms are not fully understood. However, extensive research has been completed in understanding an overview of oocyte reprogramming and epigenetic modification. The introduction of a somatic nucleus into the oocyte causes rapid deacetylation of histones on lysine residues, catalysed by histone deacetlase. Moreover, the donor chromatins also experience demethylation [12], which is also a method that is used to dedifferentiate the nuclei back to totipotent state. Aberrant or incomplete DNA reprogramming is thought to be a major contributor to abnormal development in embryos and clones which can explain why only 1% of SCNT are successful in producing fully developed clones. Figure.2The efficiency of the Honolulu technique and the success rate of cloning have been shown to be superior to the Roslin technique [12]. However, the overall success rate of cloning, irrespective of the method used is still considerably low, with only 1% success rate. Figure. 2 [13] shows the percentage of embryos surviving prior to implantation with surrogate and post implantation. Moreover, there are several risks associated with clones derived from mammalian SCNT. These risks also have ethical implications that follow. Phenotypic abnormalities that are associated with clones derived from SCNT ranges from aberrant telomere length (which can lead to premature ageing) to large offspring syndrome and irregular placenta development during embryonic growth. The telomere length and ageing of clones are thought to be directly correlated. Telomeres are situated on the ends of chromosomes and consist of numerous repetitive DNA bases that function to stabilise and prevent deterioration of the chromosome [14]. Experimental observations show that some species of mammals are prone to shorter telomere lengths in comparison with a control. It is also thought that the telomeres are not fully restored to the original length during SCNT. Such implications can suggest that the sizes of the somatic cell telomeres are inherited by the clones; therefore producing clones that have already aged [15]. Dolly lived until she was 6 years of age (half the age of an average sheep) and was shown to have shorter telomeres in comparison to a control (19 kb vs. 23 kb) implying that she died prematurely. However, shorter telomeres in clones are not universally applicable as in mice, bovine and cattle all showed similar lengths to their respective control, if not lon ger [16]. The occurrence of shorter telomere lengths in some species suggests that the donor cell species and genetic background govern it. Nevertheless, the exact cause of short telomere length is still not yet fully comprehendible, yet some studies indicate that it might be caused by incomplete reprogramming [17]. Large offspring syndrome (LOS) is characterised by larger than normal clones that have oversized organs and aberrant limb formation which all can lead to an increase in prevalence of organ defects and cardiovascular difficulties. These characteristics have been observed in cattle and can contribute to higher abortions rate and deformities in skeletal structure. However, offspringà ¢Ã¢â€š ¬Ã¢â€ž ¢s derived from cloned mammals diagnosed with LOS, were shown not to have LOS [18]. This suggests that again irregular epigenetic reprogramming during SCNT is a contributor to LOS as the progeny of the clones (which are born naturally) fail to have LOS. Embryos that are derived from SCNT have been shown to have abnormal/enlarged placenta development (placentomegaly) during embryonic growth. The abnormalities occur in both bovine and mice [19] and can cause the developing fetus to die during pregnancy. The aberrant placenta in mice is shown to have an increased amount of insulin- like growth factor which can cause LOS in clones. Moreover, failure for the placenta to develop accordingly during the pregnancy of clones can cause immune-mediated abortion [20]. The risks to mammalian reproduction stated above can produce clones that are phenotypically defective which raises ethical concerns. The abnormalities in clones can cause harmful side effects and can lead to cloned mammals suffering. We have seen that some mammals show premature ageing which can ultimately lead to premature death. The welfare of these clones seems to be disregarded in the experiments that are conducted. Moreover, there are concerns that a small proportion of cloned animals can enter our food chain, which is thought to be unsafe. However, recent studies show that consumption of cloned animals is safe to homosapeins [21]. The prospect of human SCNT also has deep ethical implications. Current legislation in all countries prevents SCNT in humans. Nonetheless, the proposed benefits that SCNT offers (therapeutic cloning) may one day outweigh the ethical concerns. If this occurs, it would shake the foundations of tradition, as humans can be à ¢Ã¢â€š ¬Ã¢â€ž ¢producedà ¢Ã¢â€š ¬Ã¢â€ž ¢ asexually with their genomic sequence known [22]. This can lead to à ¢Ã¢â€š ¬Ã‹Å"gene discrimination by other non cloned humans, and by cooperate companies who can prevent human clones (that may be prone to specific dieses) from obtaining insurance, for example. In conclusion, Somatic cell nuclear transfer has been successfully used to clone mammals from fully differentiated somatic cell. However, this technique is largely inefficient and a major Impediment is that only 1% of somatic cells successfully developed into clone. The lack of understanding on oocyte reprogramming can be contributed to the inefficiency of this technique. Moreover, this has lead to some clones showing abnormal phenotypic features which has major ethical implications. Nevertheless, somatic cell nuclear transfer shows great promise in the fields of medical therapeutics, agriculture and conservation once all aspects of its process are understood.