| Most local pig breeds in China have excellent meat quality,but excessive fat deposition leads to poor market competitiveness.Therefore,it is urgent to improve local pig breeds.Lipid droplets are the main organelles for storing fat in cells,and the size of Lipid droplets determines the cell’s ability to store fat.Thus,studying the molecular mechanism that regulates Lipid droplets size is of great significance for improving pork production traits in pigs.Perilipin 2(PLIN2)is one of the most important regulators of lipid accumulation,which promotes the formation and stability of Lipid droplets,and inhibits the breakdown of major lipids,such as triglycerides and cholesterol esters,in Lipid droplets.In this study,we used PLIN2 knockout cell lines as a model to investigate the effect of PLIN2 on Lipid droplets size in pig kidney cells,employing RNA-seq,functional acquisition and loss,transmission electron microscopy,protein immunoblotting,and real-time fluorescence quantification.The main results of this study are as follows:1.To determine the difference in lipid droplet size in local and foreign pig breeds,BODIPY staining was used to detect lipid droplets in both types,revealing that the average size of lipid droplets in Ningxiang pigs’ back fat tissue is about 5times larger than in Duroc pigs,indicating a difference in adipocyte size between Chinese and foreign pig breeds.2.Through protein immunoblotting,PLIN2 expression was measured in the back fat tissue of both local and foreign pig breeds.The results showed that PLIN2 relative expression in Ningxiang pigs was about 2 times higher than in Duroc pigs,suggesting that PLIN2 may be a key gene affecting differences in fat deposition between Chinese and foreign pig breeds.3.The PLIN2 knockout cell line was generated using dual sg RNA mediated cleavage reaction,and the sequencing and protein immunoblotting results confirmed that the target sequence was successfully knocked out in the cell line,which didn’t express at the protein level,indicating the successful acquisition of a PLIN2 homozygous knockout cell line.4.The PLIN2 knockout cell line showed a 70% decrease in lipid droplet content compared to the wild type.To confirm whether PLIN2 affects the size of lipid droplets in adipocyte precursors,si RNA was used to interfere with the expression of PLIN2 for 72 hours,and the results of BODIPY staining showed a reduction in lipid droplet content of about 30%,suggesting that stable knockout and transient silencing can both affect lipid droplet content.5.To determine the effect of PLIN2 on lipid metabolism physiology,a comprehensive quantitative lipidomics analysis was performed,showing that the deletion of PLIN2 led to about a 30% reduction in the content of major neutral lipids such as triglycerides,suggesting that PLIN2 regulates triglyceride synthesis.6.Transcriptome data analysis revealed CPT1 C as a differentially expressed gene,and its protein expression level and m RNA abundance were significantly reduced after PLIN2 knockout or si RNA interference.Conversely,the overexpression of CPT1 C led to a significant increase in PLIN2 m RNA abundance,suggesting that PLIN2 may interact with CPT1 C to regulate the size of lipid droplets.7.To demonstrate the effect of PLIN2 knockout on organismal fat deposition at the individual level,PLIN2 knockout cell line was used as donor cells for somatic cell nuclear transfer to successfully generate three PLIN2 knockout pigs.In summary,this study successfully obtained a PLIN2 knockout cell line.In summary,this study successfully obtained a PLIN2 knockout cell line,preliminarily screened differential expressed gene CPT1 C,which may interact with PLIN2 to regulate the size of lipid droplets,and successfully generated three PLIN2 knockout pigs through somatic cell nuclear transfer.The research results provide material for revealing the molecular mechanism of PLIN2’s regulation of lipid droplet size at the individual level,and provide a theoretical basis for improving meat production traits in pigs. |
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