Han Liqiang, Zhang Menglu, Xing Zhiyang, Coleman Danielle N, Liang Yusheng, Loor Juan J, Yang Guoyu
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002 PR China.
Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801 USA.
J Anim Sci Biotechnol. 2020 Jul 3;11:72. doi: 10.1186/s40104-020-00479-6. eCollection 2020.
Milk lipids originate from cytoplasmic lipid droplets (LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1A1 (BTN1A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known.
The objective was to knockout BTN1A1 in bovine mammary epithelial cells (BMEC) via the CRISPR/Cas9 system and evaluate LD formation, abundance of lipogenic enzymes, and content of cell membrane phospholipid (PL) species. Average LD diameter was determined via Oil Red O staining, and profiling of cell membrane phospholipid species via liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Lentivirus-mediated infection of the Cas9/sgRNA expression vector into BMEC resulted in production of a homozygous clone . The LD size and content decreased following gene knockout. The mRNA abundance of fatty acid synthase () and peroxisome proliferator-activated receptor-gamma () was downregulated in the clone. Subcellular analyses indicated that BTN1A1 and LD were co-localized in the cytoplasm gene knockout increased the percentage of phosphatidylethanolamine (PE) and decreased phosphatidylcholine (PC), which resulted in a lower PC/PE ratio.
Results suggest that plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.
乳脂源自细胞质脂滴（LD），其通过独特的膜包裹过程由乳腺上皮细胞合成并分泌。嗜乳脂蛋白1A1（BTN1A1）是包围脂滴的膜蛋白之一，但其在牛乳腺脂滴合成和分泌中的作用尚不清楚。
目的是通过CRISPR/Cas9系统敲除牛乳腺上皮细胞（BMEC）中的BTN1A1，并评估脂滴形成、生脂酶丰度和细胞膜磷脂（PL）种类的含量。通过油红O染色测定平均脂滴直径，并通过液相色谱 - 串联质谱（LC-MS/MS）分析细胞膜磷脂种类。
慢病毒介导的Cas9/sgRNA表达载体感染BMEC导致产生纯合克隆。基因敲除后脂滴大小和含量降低。脂肪酸合酶（）和过氧化物酶体增殖物激活受体γ（）的mRNA丰度在克隆中下调。亚细胞分析表明，BTN1A1和脂滴共定位于细胞质中，基因敲除增加了磷脂酰乙醇胺（PE）的百分比并降低了磷脂酰胆碱（PC），导致PC/PE比值降低。
结果表明，通过涉及膜磷脂组成的机制在调节脂滴合成中起重要作用。
