Abstract
BackgroundTraditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Therefore, we tried to derive goat iPSCs with a new method by transfecting exogenous Oct4, Sox2, Klf4 and c-Myc mRNAs into goat embryonic fibroblasts (GEFs), and explore the mechanisms regarding the transcription regulation of the reprogramming factors in goat iPSCs induction.ResultsmRNAs of the four reprogramming factors were transfected into GEFs, and were localized in nucleus with approximately 90% transfection efficiency. After five consecutive transfections, GEFs tended to aggregate by day 10. Clones appeared on day 15–18, and typical embryonic stem cell -like clones formed on day 20. One thousand AKP staining positive clones were achieved in 104 GEFs, with approximately 1.0% induction efficiency. Immunofluorescence staining and qRT-PCR detection of the ESCs markers confirmed the properties of the goat iPSCs. The achieved goat iPSCs could be cultured to 22nd passage, which showed normal karyotype. The goat iPSCs were able to differentiate into embryoid bodies with three germ layers. qRT-PCR and western blot showed activated endogenous pluripotent factors expression in the later phase of mRNA-induced goat iPSCs induction. Epigenetic analysis of the endogenous pluripotent gene Nanog revealed its demethylation status in derived goat iPSCs. Core promoter regions of the four reprogramming factors were determined. Transcription factor binding sites, including Elf-1, AP-2, SP1, C/EBP and MZF1, were identified to be functional in the core promoter regions of these reprogramming genes. Demethylation and deacetylation of the promoters enhanced their transcription activities.ConclusionsWe successfully generated goat iPSCs by transfection of Oct4, Sox2, Klf4 and c-Myc mRNAs into GEFs, which initiated the endogenous reprogramming network and altered the methylation status of pluripotent genes. Core promoter regions and functional transcription binding sites of the four reprogramming genes were identified. Epigenetic regulation was revealed to participate in mRNA induced iPSCs formation. Our study provides a safe and efficient approach for goat. iPSCs generation.
Highlights
Traditional approaches for generating goat pluripotent stem cells suffer from complexity and low preparation efficiency
The results above imply that mRNAs of the reprogramming factors can be transfected into goat embryonic fibroblasts (GEFs) and exert function in the nucleus, and induce GEFs into Induced pluripotent stem cells (iPSCs)
The expression of Embryonic stem cells (ESCs) markers of goat iPSCs was further confirmed by qRTPCR, and the results showed that ESCs marker genes
Summary
Traditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Isolation and cultivation of goat ESCs have been achieved over the past several decades, goat ESC lines have not been established, which largely limited the application of goat ESCs in genetic epidemiology, disease models, neomorphs, and animal species breeding. The traditional method of establishing an iPS cell line is to transfect inducible factors such as Oct, Sox, KIf4, and c-Myc into somatic cells carried by viral vectors. The efficiency of this transfection approach and, the frequency with which all of the inducible factors express simultaneously are very low. Alternative methods using plasmid vectors [13], multiple protein expression [14], small molecules [15], and transposition reduced the integration of exogenous genetic material and improved the security of iPS acquisition [16], these methods suffer from complexity and low preparation efficiency
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