Nanog
The paper describes how modern molecular biology methods are applied to identify a new protein called Nanog. They found the sequence of this new protein and determined that it is directly involved in the controlling mechanisms for self-renewal and differentiation of embryonic stem cells. The authors were able to demonstrate that Nanog, as opposed to other previously described proteins that are also involved in self-renewal, works independently an in the absence of external factors like cytokines. Their results also showed that Nanog is not always being produced in the cell; its production is restricted to the events of cell generation. Additionally, the experimental evidence suggests that nanog is abundantly expressed in gonad cells. Perhaps the main conclusion is that even in the presence of compounds and factors that are known to promote differentiation, nanog has the ability to stop the process by a mechanism that was not previously described in the available literature.
Nanog was identified by creating an extensive library of complementary DNA (cDNA). This was accomplished by transcribing, with the aid of the reverse transcriptase, several messenger RNAs into their corresponding DNA. All the transcripts were then incorporated into vectors, which were subsequently introduced via electroporation into embryonic cell lines, in a process known as transfection. To rule out the possibility of other genes being responsible for increased levels of undifferentiated cells, they were deleted from the cell lines. A selection marker within the vector allowed knowing whether the genetic information was successfully incorporated into the cells. Only cells with the phenotype of interest were collected, in this case, undifferentiated cells. The process was repeated multiple times to assure that only genetically-altered cells were considered. The vectors within the selected organisms were then sequenced to determine the genetic information that encoded for nanog. The genetic information was then transferred to more potent vectors, known as episomes, which allowed high levels of transfection.
Pluripotency is the ability of cells to remain undifferentiated. To determine that nanog was involved in the processes sustaining this ability, its expression was regulated by using the espisomes. High levels of expression demonstrated resilience to differentiation while low levels worked in the opposite direction. Further experiments were conducted to determine whether nanog works alone or with other protein partners. In that case, nanog was co-expressed with Erk, Oct4, and Stat3, which have been previously described as major participants of pluripotency. The results strongly suggested that nanog was an independent player.
