Nuclear transfer, nuclear reprogramming and the delivery of exogenous macromolecules into living amphibian cells

This thesis describes a number of foundation studies regarding Xenopus nuclear transfer, inter-species transcriptional reprogramming and a method for delivering exogenous macromolecules into living amphibian cells. I divided my research into four stages. First, I demonstrated that morphologically and reproductively normal cloned animals could be obtained following Xenopus nuclear transfer using streptolysin permeabilised donor cells. The use of streptolysin, as opposed to the traditional cell-squashing method, permits more consistent, more controlled and gentler donor cell permeabilisation, and makes nuclear transfer quicker and technically easier to perform. Second, I demonstrated that epigenetically aberrant and developmentally defective cloned Xenopus embryos possess cells that retain the capacity to differentiate into multiple cell types, survive for an extended period of time and exhibit a normal growth morphology. Third, I demonstrated that Xenopus oocytes could transcriptionally reprogram mammalian somatic cell nuclei to express Oct-4, a mammalian stem cell/pluripotency marker. Finally, I demonstrated that the reversible streptolysin permeabilisation technique, previously described for mammalian cell lines, could be modified slightly and then used with an amphibian cell line to deliver exogenous macromolecules into living amphibian cells. This modified cell delivery technique has a wide variety of potential reprogramming, transgenesis and differentiation applications for researchers working with amphibian cells. In my opinion, the long-term objectives of the reprogramming/cloning field are to identify the reprogramming molecular mechanisms, to improve the efficiency of nuclear transfer and to obtain therapeutically useful isogenic human embryonic stem cells. My research provides a number of foundation result on which these larger problems can be addressed in the future. First, my result with developmentally defective cloned Xenopus embryos provides a strong case for future research to investigate whether isogenic embryonic stem cells can be obtained from developmentally defective cloned primate/human embryos; stem cells that could potentially be used to treat various degenerative diseases.EThOS - Electronic Theses Online ServiceGBUnited Kingdo