Gene regulation
Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector. In this lab, 4 transcription factor genes were induced into fibroblast cells. Which virus did you use today?
3. in this lab, transduction was performed using a lentivirus. Member of the Retroviridae family, it is a group of viruses that carry their genetic blueprint as an RNA genome.
Why do we need to analyze these samples as well? (RT-PCR results from embryonic stem cells and fibroblast cells)
Because those samples are the experimental controls in this case, embryonic stem cells are positive control because they express the NANOG gene and fibroblast cells are negative control because they do not express NANOG
This is the result of the western blot. Which cells do not express NANOG protein?
Cells that have been transduced with general transcription factors the positive control (ES cells) and the cells transduced with unique TF express NANOG, the cells transduced with general TF and the negative control (fibroblast cells) don't express NANOG protein
Examine the RT-PCR result that shows gene expression of NANOG in the cells. Which cells express NANOG?
Cells that have been transduced with unique transcription factors both cell-unique TF and embryonic stem cells express NANOG
How do we obtain a protein sample from cells?
Lyse the cells Cell lysis breaks the barrier to the extra cellular environment (enclosed by plasma membrane) and allows us to obtain the proteins inside the cells.
What kind of regulation is the phosphorylation and degradation by proteasomes?
Post-translational eukaryotic polypeptides often undergo post-translational modifications. These modifications can either prolong, decrease the half-life of a protein thus regulating the activity in the cell
What type of cells do you have on this plate?
Stem cells
The gene expression of NANOG is up regulated in iPS cells but silenced in differentiated cells. What does this suggest about NANOG?
Sustains stem cells self renewal ability it encodes a protein which has the ability to sustain stem cell self-renewal and is important for maintaining pluripotent characteristics
Look at the screen next to the microscope to examine the morphology of the cell lines. Which cell exhibits stem-like morphology?
The one that was transduced with the unique transcription factors However, cells that have been transduced with general transcription factors exhibit fibroblast-like morphology.
Why is it important to also analyze the NANOG protein level in the cells?
To monitor post-transcriptional modifications! since proteins are the functional product of protein coding gene's expression, analyzing protein levels is the final proof of effective gene expression. 1 explanation for the discrepancy between mRNA and protein levels are the post transcriptional modifications
In eukaryotes a combination of molecules and region of DNA will increase the gene expression rate, which?
Transcription factors and an enhanced region
The diagram above shows a eukaryotic cell where the central dogma...
Transcription, premRNA, mRNA, ribosome
Do the results confirms your initial hypothesis, that the unique transcription factors induce pluripotency?
Yes, they show characteristics of stem cells from the cell morphology analysis we can see that the cells transduced with unique transcription factors resemble a stem cell. From mRNA and protein level analysis we know that these cells express NANOG, a gene that is involved in maintaining pluripotency in stem cells. These results indicate that the unique transcription factors that we selected could induce pluripotency by altering gene expression (same thing Shinya Yamanaka demonstrated)
Why are induced pluripotent stem cells used instead of embryonic stem cells?
iPS are better to avoid immunological reactions the ES -they will not trigger immunological reactions that result in transplant rejections
