A High-throughput Screening Method For Genes Associated With Reprograming By PiggyBac Gene Trap System

Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. Just like embryonic stem cells (ESCs) iPSCs can propagate indefinitely, as well as give rise to every other cell type in the body, so they hold great promise in the field of regenerative medicine, disease modeling and drug development. Shinya Yamanaka’s lab in Japan that successfully converted adult cells into pluripotent stem cells in 2006 developed an assay system that screened four specific transcription factors from 24 factors. So far scientists have discovered more reprogramming factors such as Nr5a2 that can replace the dominative factor Oct4 and new four factors combination that is Sall4, Esrrb, Lin28, Dppa2 or Nanog. The miRNA, protein or small molecules can even reprogram adult cells. The opinion that the four defined transcription factors can’t be replaced was subverted. We gained more insights into the process of reprogramming. The more reprogramming factors are found, the more deepened we understand the reprogramming mechanism. Therefore we established a new simple system that could screen comprehensively genes related to reprogramming in genome wide.Based on piggyBac gene-trap system and next generation sequencing technology we generated a forward genetics screening method that could rapidly confirm mutation site.We manufactured PB transposon plasmid into which the three mouse factors (OKS or KSM) were transferred and we also generated the piggyBac gene-trap vector. We electroporated gene-trap vector, the three mouse factor vector and PBase into Oct4-GFP MEFs and then picked GFP+colonies. We confirmed insertion site to find the reprogramming related genes by inverse-PCR.By analysis 300 colonies we found many well-known reprogramming factors or related family genes such as Nr6a1, Nr5a1, Klf5, Smarcadl, Sox2, Mycbp2, Mir205 and so on. This experiment demonstrated the screen system was available to cell reprogramming. Afterwards, we analyzed 500 thousand colonies. Mutation heat map and a genes list ranking by reprograming capability were presented. We analysis the gene fuctions and signal pathways. The top 1000 genes distributed into KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways mostly related to innate immunity such as Cytosolic DNA-sensing pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and so on. Then the 1000 genes partially related to pathways in cancer and some well-known reprogramming related signaling pathways such as JAK-STAT signaling pathway, Notch signaling pathway and so on. We gained more insights in reprogramming that innate immunity pathways may play an important role in cell reprogramming.In the heat map the hottest region was IFN gene locus. In the process of cell programming we treated the cell culture with IFN of various sources. We observed that human IFNalb promoted human fibroblast cells reprogramming. Likewise,50U/ml mouse IFNa can enhance MEFs reprogramming by 25%. The efficiency of MEFs reprogramming was increased 2 times by 200U/ml mouse IFNy. The results exhibited dosage effect like LIF, that is, IFN promoted cell reprogramming only in appropriate concentration. It is indicated that the genome-wide screening method was useful.