[Preparation of in vitro transcribed mouse MUC1 mRNA and its expression in NIH/3T3 cells]

Objective To obtain in vitro transcription of mouse mucin 1(MUC1) mRNA and express it in NIH/3T3 cells. Methods The MUC1 gene was amplified from mouse 4T1 cells by reverse transcription PCR (RT-PCR), and then inserted into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant vector pcDNA3.1(+)-MUC1. After identification by restriction enzyme cutting, the recombinant plasmid was used as a PCR template for the amplification of the MUC1-HA TAG fusion gene fragment with the reverse primer containing hemagglutinin tag (HA TAG) sequence. Subsequently, the MUC1-HA TAG fusion gene was cloned into the expression vector pcDNA3.1(+). After double restriction enzyme digestion and DNA sequence identification, the recombinant plasmid encoding MUC1-HA TAG was used as a PCR template for the amplification of a PCR product containing T7 promoter and the MUC1-HA TAG fusion gene fragment as an in vitro transcription template to obtain the modified MUC1-HA TAG mRNA by in vitro transcription, tailing and purification. The resulting MUC1-HA TAG mRNA was transfected into NIH/3T3 cells by various transfection reagents and the expression of the fusion protein MUC1-HA TAG was detected by Western blot analysis. Results The MUC1 gene amplified by RT-PCR was about 1 900 bp in length. The restriction enzyme digestion and sequence analysis confirmed that the MUC1-HA TAG fusion gene had been successfully inserted into pcDNA3.1(+), and the insertion sequence was identical to the MUC1 gene sequence of the mouse in GenBank database. HA TAG was correctly inserted in the downstream of MUC1 gene and the reading frame was correct. Western blot analysis indicated that the in vitro transcriptionally modified MUC1-HA TAG mRNA could be expressed in NIH/3T3 cells. Conclusion In vitro transcriptionally modified mouse MUC1-HA TAG mRNA can be translated and expressed in mammalian NIH/3T3 cells.