An important question that was asked by early developmental biologists was, is cellular differentiation due to the loss of genetic information? Is the genetic complement of a neuron different from a skin cell or a muscle cell? This question was first approached by Briggs and King in the 1950s through nuclear transfer experiments in frogs. These experiments were extended by Gurdon and McKinnell in the early 1960s320. They were able to generate adult frogs via nuclear transfer using embryonic cells. The process was inefficient however - only a small percentage of transferred nuclei (taken from differentiated cells) supported normal embryonic development. Nevertheless, these experiments suggested that it was the regulation rather than the loss of genetic information that was important in embryonic differentiation.
In 1996 Wilmut et al used a similar method to clone the first mammal, the sheep Dolly. Since then many different species of mammal have been cloned, and there is serious debate about the cloning of humans. In 2004, cloned mice were derived from the nuclei of olfactory neurons using a method similar to that used by Gurdon. These neurons came from a genetically engineered mouse that expressed GFP (see above). A hybrid gene contained the coding sequence for GFP and a regulatory sequence that led to its expression in most cell types of the mouse. Neuronal nuclei were transplanted into an oocyte from which the original nucleus had been removed (an enucleated oocyte). Blastula derived from these cells were then used to generate totipotent embryonic stem cells. It was the nuclei from these cells that were transplanted into enucleated eggs. The resulting embryos were able to develop into full grown and fluorescent mice, proving that neuronal nuclei retained all of the information required to generate a complete adult animal.
The process of cloning from somatic cells is inefficient – many attempts have to be performed, each using an egg, to generate an embryo that is apparently normal (most embryos produced this way were abnormal). At the same time, there are strong ethical concerns about the entire process of reproductive cloning. For example the types of cells used, embryonic stem cells, are derived from the inner cell mass of mouse or human embryos. Embryonic stem cells can be cultured in vitro and under certain conditions can be induced to differentiate into various cell types. Since the generation of totipotent human embryonic stem cells involves the destruction of a human embryo, it raises a number of ethical issues.
Current research attempts to avoid these issues by focussing on optimizing the process by which somatic nuclei can be reprogrammed to support totipotent and pluripotent development. In this scenario, somatic cells from a patient are treated with genes (or more recently gene products) of a small number (typically) four molecules to induce differentiated somatic cells to become pluripotent cells. These “induced pluripotent stem cells” (iPSCs) behave much like embryonic stem cells. The hope is that iPSCs derived from a patient could be used to generate tissues or even organs that could be transplanted back into the patient, and so reverse and repair disease-associated damage.
Questions to answer & to ponder:
- How might asymmetries be generated in the zygote?
- How could two cells that express the same set of transcription factors, express different genes?
- In terms of transcription factors and chromatin packing, why is it difficult to reverse differentiation?
- Why might the organism want to reduce the number of stem cells it contains?
- Based on your understanding of the control of gene expression, outline the steps required to reprogram a nucleus so that it might be able to support embryonic development.
- What is necessary for cells to become different from one another - for example how do muscle cells and skin cells come to be different from one another?
- What are the main ethical objections to human cloning? What if the clone were designed to lack a brain, and destined to be used for "spare parts"?
320 The egg and the nucleus: a battle for supremacy: http://www.nobelprize.org/mediaplayer/?id=1864