pLVX-TetOne-Puro

Mammary epithelial cells are the only cells in the mammary gland that have the ability to produce milk. They are closely related to breast development and milk production. Early studies have shown that the transformation of goat fibroblasts into induced mammary epithelial cells (iMEC) is closely related to the overexpression of SMAD3. In this study, the role of SMAD3 in iMEC reprogramming was further explored. The SMAD3 gene was overexpressed in goat ear fibroblasts using a tetracycline-induced expression method. The results show that goat ear fibroblasts can be converted into iMECs by overexpressing the SMAD3 gene. In contrast, the researchers found that downregulation of SMAD3 by RNA interference significantly reduced iMEC reprogramming efficiency. These results indicate that SMAD3 plays a critical regulatory role in iMEC reprogramming.

In this study, in order to explore the function of goat SMAD3 gene in inducing mammary epithelial cell reprogramming, the researchers regulated the expression level of SMAD3 gene in goat fibroblasts. Primers were designed using goat SMAD family member 3 (SMAD3) mRNA sequence, pUC57 2As, and pLVX-IRES-ZsGreen1 plasmids. SMAD3, P2A, and ZsGreen1 fragments carrying homology arms were amplified by RT-PCR and spliced into SMAD3-P2A-ZsGreen1 by overlap extension PCR (SOE PCR). SMAD3-P2A-ZsGreen1 was inserted into the EcoRI/BamHI sites of pLVX-TetOne-Puro (pLVX; empty vector). Then, doxycycline (Dox)-induced overexpression of SMAD3 lentiviral plasmid (pLVX-TetOne-SMAD3-P2A-ZsGreen1-Puro) was constructed. Expression of the P2A-linked green fluorescent protein ZsGreen1 in this vector allowed researchers to estimate SMAD3 expression in an approximate manner. The eukaryotic expression vector pSicoR-Ef1a-mCherry-shRNA-SMAD3 was also obtained, which targets the SMAD3 gene in goats.

Figure 1. Agarose gel electrophoresis analysis of overexpression and construction of eukaryotic expression vectors for the interfering goat SMAD3 gene. (Wu Y, et al., 2022)