Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska, 1083, 142 20, Prague 4, Czechia.
Transgenic Res. 2017 Oct;26(5):689-701. doi: 10.1007/s11248-017-0027-0. Epub 2017 Jun 20.
Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.
富含亮氨酸重复序列的 G 蛋白偶联受体 4（LGR4）在广泛的小鼠胚胎和成年组织中产生，其缺乏导致胚胎或围产期致死。LGR4 的功能主要与其增强经典 Wnt 信号有关；然而，最近的几项研究将 LGR4 与其他信号通路联系起来。为了获得研究 Lgr4 信号特性的合适工具，我们使用小鼠卵母细胞中的基因靶向生成了 Lgr4 受体的标记变体。该修饰的 Lgr4 等位基因表达与位于蛋白质胞外部分的三聚体血凝素 (3HA) 标签融合的 Lgr4 蛋白。该等位基因具有完全的功能，能够在小鼠组织中跟踪 Lgr4 的表达。我们还表明，通过表面标记，3HA 标签允许直接分离和分析从小肠隐窝获得的活的 Lgr4 阳性细胞。最后，HA 标签特异性抗体可用于表征 Lgr4 的生化特征，并鉴定来自各种小鼠组织的细胞中该蛋白的可能结合伙伴。
