Validating the effect of Rlf and Scml2 on somatic cell reprogramming (360G-Wellcome-207245_Z_17_Z)

Oct4, Sox2 and Nanog (OSN) are important pluripotency transcription factors that play an important role in embryonic stem (ES) cell self-renewal. Oct4 and Sox2 are also key factors in reprogramming somatic cells into induced pluripotent stem (iPS) cells. In a recent study, proteins interacting with OSN on chromatin were identified by chromatin immunoprecipitation followed by Mass spectrometry named ChIP-SICAP (Rafiee, et al, 2016). Independently, the Kaji lab performed a genome-wide knockout screening using a guide RNA (gRNA) library of 90,000 gRNAs to identify detrimental and essential genes for reprogramming. The screening data indicated that knockout of little characterized OSN-associated protein, Rlf or Scml2, decreases or increases reprogramming efficiency, respectively, while knockout of neither gene demonstrated obvious phenotype in ES cell self-renewal. The goal of the project is to evaluate the effects of knockout and overexpression of Rlf or Scml2 on reprogramming. For this purpose, I will make vectors which can express reprogramming factors and gRNA or cDNA simultaneously, and perform reprogramming. By counting resulting iPSC colony numbers and comparing with a control condition which does not express Rlf or Scml2 gRNA/cDNA, I will investigate roles of Rlf and Scml2 in reprogramming.