Date of Completion
Animal Sciences | Cell Biology | Other Cell and Developmental Biology
Embryonic stem cells are unique in their ability to grow indefinitely while maintaining both pluripotency and self-renewal capabilities. These stem cells could be the answer to solving many of the debilitating and devastating diseases suffered by humans. Recently, the use of human derived embryonic stem cells has been effective in animal models in the treatment of spinal cord injuries (Kierstead et al., 2005).
Although the study of embryonic stem cells has become increasingly popular in the last decade, there is still much research to be done to increase the expression of valuable embryonic proteins which will induce pluripotency in the cell. These proteins have been found to reprogram murine somatic cells into induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006). Research conducted in the last decade has revealed four transcription factors which are vital to the induction of pluripotency in differentiated cells. iPS cells are derived using these purified proteins through protein transfection.
This study focused on four of these transcription factors, Oct4, Sox2, Myc, and Klf4. Although each of these factors has different functions many of them act synergistically to promote pluripotency and self-renewal. To increase the expression of these transcription factors by a host cell, Escherichia coli, genes for the chaperone protein and transcription factors are induced using combinations of tetracycline and L-arabinose.
Chaperone plasmids increase protein folding and the ability of the host to express foreign DNA. The purpose of this study was to combine the four transcription factors with one of the two chaperone plasmids, pG-Tf2 and
pG-KJE8 which have been given different inducers to determine which combination for each transcription factor has the highest expression. In order to determine this, bacterial host cell colonies were raised to contain one of the four factors. Each colony was raised with one of the two chaperone plasmids. Colonies which contained the pG-Tf2 plasmid were induced using tetracycline only since they only contained one promoter region. Colonies containing the pG-KJE8 plasmid were induced with either tetracycline, L-arabinose or both since it contains two promoter regions.
When the pG-Tf2 chaperone plasmid was used there was positive expression in the Sox2 colonies but no expression was seen in the soluble fraction of any other colony. pG-KJE8 induced with only tetracycline had no expression in any colony. Induction with L-arabinose produced a positive result in the Sox2 and Oct4 colonies. When pG-KJE8 was induced with tetracycline and L-arabinose there was no expression in the soluble fraction from any colony. None of the four conditions tested provided any response in the soluble fraction from Myc or Klf4 indicating the need for further research on the best conditions for expression of these proteins.
Wilson, Kaitlin, "Optimization of Protein Expression of Four Transcription Factors Using Chaperone Proteins" (2009). Honors Scholar Theses. 68.