Woltjen Lab

Stem Cells and Genome Engineering

Written by: on March 12, 2015 @ 16:03

Terminator

Dr. Shin-Il Kim and his lab mates have been working like machines to unravel a reprogramming mystery. Employing piggyBac transposons to deliver two seemingly similar polycistronic reprogramming cassettes (both employing the Yamanaka Factors Oct3/4, Sox2, Klf4, and c-Myc), achieves two drastically distinct reprogramming outcomes. With one vector, colonies are plentiful, yet ultimately fail to fully reprogram. With the other, colonies are few but true – with most going on to activate a Nanog-GFP pluripotency reporter.

So, why the difference? Looking down to the sequence level, we found that the Klf4 open reading frame in the two vectors differed subtly in length. In the vector which produced primarily partially reprogrammed cells, Klf4 was truncated by 9 N-terminal amino acids. Amazingly, replacement of these 9 amino acids suppressed colony formation and recovered the reprogramming capacity of the deficient vector.

These phenotypes are ultimately achieved by modulating the relative factor stoichiometry, where short Klf4 produces less protein than long. Interestingly, this affect appears to be dependent on 2A-peptide linkage, as monocistronic short and long Klf4 function nearly indistinguishably. Importantly, this modulation of stoichiometry is reflected in global gene expression changes during reprogramming, highlighting difficulties in direct comparison of gene expression profiles from distinct reprogramming systems.

Read more about our findings on the CiRA Website CiRA Website (日本語 or English), on EurekAlerts, or jump directly to the Stem Cell Reports Resource Article!

This work was made possible through coordinated efforts of all our lab members, and the invaluable help of our collaborators in the Yamada, Yamamoto, and Yamanaka Labs at CiRA. Our reprogramming research is supported by funding from the Funding Program for World-Leading Innovative R&D on Science and Technology Program of the Japan Society for the Promotion of Science (FIRST, JSPS) and the Strategic International Collaborative Research Program of the Japan Science and Technology Agency (SICORP, JST).

Photo copyright of the Terminator franchise – Megan Ellison / James Cameron

Categories: Publications