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siRNA transfection and shRNA transduction optimization for CXCL12 mRNA silencing in human bone marrow mesenchymal stem cells
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  • Sabrina do Nascimento,
  • Fernanda Mansur,
  • Fernanda Rocha,
  • Luciana Marti
Sabrina do Nascimento
Hospital Israelita Albert Einstein
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Fernanda Mansur
Hospital Israelita Albert Einstein
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Fernanda Rocha
Hospital Israelita Albert Einstein
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Luciana Marti
Hospital Israelita Albert Einstein

Corresponding Author:[email protected]

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Abstract

RNA interference (RNAi) is a gene-silencing process elicited by double-stranded RNAs. This methodology can be used for analyzing loss-of-function phenotypes and has highly contributed for cells molecular biology research field. This study was conducted in order to optimize siRNA transfection and virus-mediated short-hairpin RNA (vshRNA) for silencing CXCL12 mRNA in human bone marrow mesenchymal stem cells (BM-hMSC) intending further studies on its role in hematopoietic stem cells (HSC) in the bone marrow niche. CXCL12 mRNA levels transient reduction was observed after siRNA transfection for silencing CXCL12 mRNA. However, there were no significant differences in intracellular CXCL12 protein levels or in cultures supernatant. On the other hand, the transduction of CXCL12-targeting vshRNA resulted in downregulation of the CXCL12 mRNA and protein levels in both intracellular compartment and culture supernatant of BM-hMSCs. Our results demonstrated very high level of transduction efficiency, without repeated rounds of infection, neither cell sorting nor antibiotic selection and with no major changes in cell viability or morphology. Thus, the current study presents an effective method for functional virus transduced BM-hMSC. In addition, this study discusses the requirement for testing candidate sequences and shRNA screening before confirming their effectiveness. Additionally, this study demonstrated that a gene reporter as green fluorescent protein (GFP) signal is not necessary closely correlated with knockdown efficiency.