An improved assay to measure the phospholipid transfer activity of microsomal triglyceride transport protein

Abstract

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Impact of Maternal Obesity on Liver Disease in the Offspring: A Comprehensive Transcriptomic Analysis and Confirmation of Results in a Murine Model

“…Genes involved in lipid metabolism were amongst the genes most consistently differentially regulated in the different datasets. For example, Mttp , a gene that encodes for microsomal triglyceride transfer protein, is upregulated in five different conditions out of ten [ 47 ]. This protein plays a central role in the lipoprotein assembly and is the target of lomitapide, an approved lipid-lowering agent for the treatment of familial hypercholesterolemia [ 48 ].…”

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

“…Human hepatoma Huh-7 cells and plasmids pcDNA3 and pcDNA3-hMTP-FLAG have been described previously ( 5 , 9 , 10 , 26 , 27 ). The MTP CRISPR/Cas9 KO plasmid containing MTP gRNAs (#SC-401884) and MTP-HDR plasmid (#SC-401884-HDR, Santa-Cruz Biotechnology) that contains sites for homologous recombination with the endogenous MTTP gene and genes encoding for red fluorescent protein (RFP) and puromycin resistance.…”

“…To knockout the MTTP gene by plasmid-based CRISPR system, plasmids expressing gRNA and CRISPR/Cas9 KO plasmids were co-transfected with MTP-HDR plasmids (1:1 ratio) into Huh-7 cells by chemical method using EndoFectin Max transfection reagent (Genecopoeia, #EF013) as described earlier ( 8 , 10 , 27 ). Huh-7 cells were seeded in T75 flasks and allowed to grow until they reached 80%–90% confluence.…”

“…pcDNA3 or pcDNA3 carrying hMTP cDNA (3 μg) were transfected using EndoFectin Max transfection reagent (Genecopoeia, #EF013). The expression of hMTP protein in wild type Huh-7 cells as well as MTP KO cells transfected with pcDNA3 carrying MTP cDNA were analyzed by MTP activity and Western blot techniques as described earlier ( 10 , 27 ). In brief, cells were lysed in buffer K by sonication with pulse of 2 s on 1 s off for 90 s and centrifuged at 12,000 rpm (13,500 g ) at 4°C for 10 min to remove unbroken cells and cell debris.…”

“…In brief, cells were lysed in buffer K by sonication with pulse of 2 s on 1 s off for 90 s and centrifuged at 12,000 rpm (13,500 g ) at 4°C for 10 min to remove unbroken cells and cell debris. The clear lysates (50 μg) were used for MTP triglyceride transfer assay as before ( 8 , 27 , 29 , 30 ). In addition, approximately 300 μg of crude protein was used for MTP purification using anti-Flag M2 affinity gel (Sigma, A2220-5ML).…”