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Transcriptome sequencing analysis of MyoD-induced transdifferentiation in 10T1/2 fibroblasts using RNA-seq

ABSTRACT: Transcriptome sequencing analysis of MyoD-induced transdifferentiation in 10T1/2 fibroblasts using RNA-seq

PROVIDER: PRJNA667738 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	SRR12782009_1.fastq.gz	Fastqsanger.gz
	SRR12782009_2.fastq.gz	Fastqsanger.gz
	SRR12782010_1.fastq.gz	Fastqsanger.gz
	SRR12782010_2.fastq.gz	Fastqsanger.gz

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Project description:Synthetic transcription factors can be applied to many areas of biotechnology, medicine, and basic research.Â Currently, the most common method for engineering synthetic transcription factors has been based on programmable DNA-binding domains of zinc finger proteins, Transcription Activator-Like Effectors (TALEs), and most recently the CRISPR/Cas9 system. These transcription factor platforms consist of the DNA-binding domain fused to potent transcriptional activation domains, most commonly the tetramer of the minimal transactivation domain of the VP16 protein from herpes simplex virus, referred to as VP64. Although many applications are well-suited for the targeted activation of a single gene, genetic reprogramming requires the coordinated regulation of many nodes of natural gene networks as is typically performed by naturally occurring reprogramming factors. Thus we sought to combine principles from each of these approaches by attaching potent transcriptional activation domains to a natural reprogramming factor to increase the efficiency and/or rate of cell fate conversion. In this study, we evaluated the effects of fusing potent activation domains to the transcription factor MyoD, the master regulator of the skeletal myoblast lineage. In certain non-myogenic lineages, MyoD overexpression causes upregulation of the myogenic gene network and conversion to a myoblast phenotype including cell fusion into multinucleated myotubes. Compared to wild-type MyoD, the VP64-MyoD fusion protein induced greater overall reprogramming of global gene expression. This simple approach for increasing the potency of natural reprogramming factors circumvents the need for screening engineered proteins and leads to a more robust cellular reprogramming compared to treatment with the wild type transcription factor. Human dermal fibroblasts were transduced with a single tet inducible lentivirus that expresses either WT-MyoD or VP64-MyoD in response to treatment with doxycycline. Untreated human dermal fibroblast served as the negative control. Gene expression was measured using mRNA-seq, and differential expression was calculated using DESeq. All experiments were performed in biological duplicates.

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Transcriptome sequencing analysis of MyoD-induced transdifferentiation in 10T1/2 fibroblasts using RNA-seq

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