Somatic Cell Cloning

Abstract

Abstract. With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.The ever-expanding gap between availability of organs and the number of patients awaiting an organ transplant is of great concern. In 2003, 14,880 transplantations were carried out between January and August, yet more than 83,000 patients were in the organ waiting list (based on data from the Organ Procurement and Transplantation Network as of November 1, 2003). This increasing divergence between availability of organ donors and the increasing number of patients in waiting lists has caused researchers to look into alternative methods to fulfill these needs. Whereas for complete organ replacement, xenotransplantation with pig organs is the main alternative, for tissue repair, other alternatives exist. The most promising to date is the use of embryo-derived and adult-derived stem cells (1). Cloning by nuclear transfer has a key role to play both by assisting in the generation of swine with complex genetic manipulations, and in the generation of human stem cells. Although ethical concerns need to be considered for the application of these technologies, such discussion is beyond the scope of this review. This review will focus on the development of the cloning technology, the basic concept in nuclear reprogramming, and recent advances in both understanding the epigenetic process of nuclear reprogramming and in enhancing the ability to undertake complex genetic manipulation in this species.The goals of generating a complete organism by nuclear transfer of a nuclei into an oocyte, or reproductive cloning, has been pursued for many years. By use of frog (Rana pipiens) eggs, Briggs and King (2) described the first nuclear transfer experiment. Briefly, ovulated MII eggs were enucleated with a handmade needle, and cells from a developing frog were lysed and their nuclei injected into the egg. Over the next 4 to 11 d, the majority of the cleaved blastulae developed into normal postneurula and tadpole stages, demonstrating the ability of the injected nuclei to at least partially complete normal de...

Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set

“…In our data set, more than half of cloned pigs were stillborn (23.6%) or died soon after birth (31.4%). Associated pathological changes, such as underweight (average weight of the cloned piglets born under 1000 g: 686.4 +/- 181.0 g; range: 375 – 973 g), which is one of the major causes of early neonatal death, or cleft palate, contracted tendons, or enlarged tongues, have also been observed by other groups [43-47]. We have the impression that the percentage of underweight piglets (among normal weight littermates) is higher in cloned litters.…”

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

“…However, compared with other reproductive assistant techniques, scNT cloning is associated with particularly high levels of phenotypic instability (Hill et al, 1999;Cibelli et al, 2002;Hiendleder et al, 2004;Park et al, 2004Park et al, , 2005, as 64% of cattle, 40% of sheep, 60% of pigs, and 93% of mice exhibit some form of abnormality, such as pulmonary hypertension and respiratory distress. For example, goat and pig clones are prone to bacterial infections of the lungs, abnormal numbers of teats, cleft lips, malformed limbs, an atrial septal defect, a cranial abnormality, and contracted tendons Piedrahita et al, 2004;Lee et al, 2005). It should also be noted that many researchers have observed that cloned animals are healthy and normal (Bondioli et al, 2001;Lanza et al, 2001;Lai et al, 2002;Yin et al, 2002).…”

39 Factors Influencing the Efficiency of Generating Genetically Engineered Pigs by Nuclear Transfer: Multi-Factorial Analysis of a Large Data Set

“…In our data set, more than half of cloned pigs were stillborn (23.6%) or died soon after birth (31.4%). Associated pathological changes, such as underweight (average weight of the cloned piglets born under 1000 g: 686.4 +/− 181.0 g; range: 375 -973 g), which is one of the major causes of early neonatal death, or cleft palate, contracted tendons, or enlarged tongues, have also been observed by other groups [43][44][45][46][47]. We have the impression that the percentage of underweight piglets (among normal weight littermates) is higher in cloned litters.…”