Endogenous Oct4Gene Activation By Artificial Transcription Factors In Mouse Embryonic Fibroblast

Targeted genome modification is a new method for study of molecular genetics andevolution, and has great potential applications in current biology. Traditional genomemodifications were mediated by mechanisms of spontaneously homologous recombination orretroviruses, which lead to low efficiency and random integartions. Modern genome targetingtools, such as TALE and CRISPR/Cas9system, not only can bring mutations at a specific site,but also generate gene knock-out, gene knock-in and gene expression regulation with highefficiency and specificity. The affinity of TALE proteins bind to targeting DNA depends onthe tandem repeat DNA-binding domain. CRISPR/Cas9system can recognize DNA throughsgRNA which guides Cas9protein to target DNA sequence. Oct4gene only expressed intotipotent cells. Oct4is the most important transcription factors in the process of somatic cellreprogramming. In this study we used TALE and CRISPR/Cas9system to target upstreamsequence of mouse Oct4gene transcription start site. We tried to activate endogenous Oct4gene expression with TALE transcription activation factor or Cas9transcription activatingfactor in mouse embryonic fibroblasts.Four TALE monomers each can recognize single deoxyribotide(A, C, T or G) wereamplified with four pairs of primers respectively. Then we constructed TALE tetramerplasmids using cut/ligation reaction based on Golden Gate shuffling strategy. We harvested alibrary contained256tetramer plasmids which covered all quadruple bases.Tetramer plasmids and artificial transcription factor backbones were used to constructTALE transcription activation factor by cut/ligation reaction. Three designed13.5-repeatTALE transcription activation factors named TALE-TF-Oct41, TALE-TF-Oct42,TALE-TF-Oct43, can target-88~-101site,-1042~-1055site,-1561~-1574site upstream ofOct4transcriptional start site, respectively.TALE-TF-Oct41, TALE-TF-Oct42and TALE-TF-Oct43plasmids were transfectedinto primay MEF with electroporation and rsulting in up-regulating Oct4gene expressionwith418-fold,2.8-fold and5-fold, respectively.In addition, three Cas9transcription activating factors and the sgRNAs were designed totarget-109~-128site,-200~-220site,-301~-322site upstream of Oct4transcriptional start site, and up-regulated Oct4gene expression with2.3-fold,1.5-fold,2.2-fold, respectively.