Eukaryotic expression vector construction, structure and function prediction and preliminary verification of mouse <italic>Klf11</italic> gene

<bold>Objective</bold> This study aims to clone the Kruppel-like transcription factor family member 11（<italic>KLF11</italic>）gene from mice，construct its eukaryotic expression vector，and investigate its regulatory effects on lipid metabolism-related genes after overexpression of Klf11 in the mouse AML12 hepatocyte cell line. <bold>Method</bold> Using cDNA from mouse liver tissue as a template，the coding sequence （CDS） of the mouse <italic>Klf11</italic> gene was amplified by PCR.The amplified fragment was then ligated into the pcDNA3.1-Puro-N-3HA vector to construct a recombinant plasmid.The recombinant plasmid was verified through restriction enzyme digestion，PCR，and sequencing，and the correct plasmid was named pcDNA3.1-mKLF11.Bioinformatics analysis of the Klf11 sequence and its encoded protein was performed using online software.Immunohistochemistry was used to detect KLF11 expression in mouse liver tissue.The pcDNA3.1-Puro-N-3HA and pcDNA3.1-mKLF11 plasmids were transfected into HEK293T and AML12 cells.The expression of the mouse <italic>Klf11</italic> gene was detected using quantitative real-time PCR （qPCR） and Western blotting.Finally，the effects of Klf11 overexpression on lipid metabolism in AML12 cells treated with free fatty acids （FFA） were assessed using qPCR and Oil Red O staining. <bold>Result</bold> The results of PCR，restriction enzyme digestion，and sequencing confirmed the successful cloning of the CDS region of the mouse Klf11 gene and the construction of the pcDNA3.1-mKLF11 recombinant plasmid.Bioinformatics analysis revealed high sequence similarity in the coding region of the <italic>Klf11</italic> gene between mice，rats，and humans.The mouse KLF11 protein exhibited hydrophilic characteristics，with its secondary structure comprising random coils，α-helices，extended strands，and β-turns.It lacked signal peptides and transmembrane regions.The tertiary structure of the mouse KLF11 protein showed significant differences compared with KLF11 proteins of humans and rats.Immunohistochemistry results indicated that KLF11 was primarily expressed in the nuclei of mouse hepatocytes.qPCR and Western blotting results showed that in both HEK293T and AML12 cells，the mRNA and protein levels of Klf11 were significantly higher in the pcDNA3.1-mKLF11 transfection group than those of the control group.Furthermore，under FFA treatment，Klf11 overexpression significantly increased the expression of lipid metabolism-related genes Fasn，Acaca，and Plin2，and increased the number of intracellular lipid droplets in AML12 cells. <bold>Conclusion</bold> This study successfully constructed an eukaryotic expression vector for the mouse <italic>Klf11</italic> gene and conducted bioinformatics analysis of the mouse <italic>Klf11</italic> gene and its encoded protein.Immunohistochemistry results show that KLF11 is primarily expressed in the nuclei of mouse hepatocytes.Additionally，KLF11 protein is successfully overexpressed in AML12 cells，and Klf11 overexpression significantly promotes lipid accumulation in AML12 cells.This research lays the groundwork for further investigation into the role of the <italic>Klf11</italic> gene in regulating lipid metabolism in mammalian livers and provides new insights for developing therapies for lipid metabolism disorders in livestock.