Preparation of Pklr gene knockout mice and study on its glycolipid metabolism

Objective Pyruvate Kinase (PK) is expressed principally in hepatocytes and red blood cells. PK is the last rate-limiting enzyme of glycolysis and plays an essential role in glycolipid metabolism. Previous studies focused on cells and clinical observations and the lack of animal models limited further studies. Thus our study aimed to generate a Pklr knockout mouse model to better understand the roles of PK in glycolipid metabolism. Methods In this study, we utilized CRISPR/Cas9 system and designed primers for sgRNA1and sgRNA2 targeting LPK exon1 and exon3 respectively. Measures including sequencing, real time quantitative PCR, Western blot and PK assay kit were taken to detect the changes in Pklr gene knockout mice on mRNA, protein expression and pyruvate kinase activity levels. Moreover, by providing normal diet (ND) and 45% high fat diet (HFD), we tested mice body weight, white adipose tissue and spleen weight to confirm the relationship between PK and glycolipid metabolism. Results Compared to the wild type mice, the absence of detectable mRNA(24%), protein expression and pyruvate kinase activity(33%) confirmed successful PK knockout. Moreover, by 45% high fat diet (HFD), the mutant homozygote mouse showed decreased in body weight and white adipose tissue(P=0.0040, 0.0200), and by normal diet (ND) and 45% high fat diet (HFD), spleen weight were increased(P=0.0003). Conclusion We have generated Pklr knockout mouse line, using the RNA-guided Cas9 nuclease gene editing system. We found the changes in abnormal glucose and fat metabolism. The Pklr gene knockout mouse model provides an platform to explore the regulatory mechanism of PK and the relationship of glucolipid metabolism in vivo. Key words: CRISPR/Cas9; Pklr; Gene knockdown; Inflammation response; Glucose and lipid metabolism