瘦素基因启动的新型脂肪细胞表达Cre工具小鼠的构建

doi:10.16288/j.yczz.22-236

遗传 ›› 2022, Vol. 44 ›› Issue (10): 950-957.doi: 10.16288/j.yczz.22-236

瘦素基因启动的新型脂肪细胞表达Cre工具小鼠的构建

曾帆(), 王澜, 万小勤, 黄荣凤, 张志辉, 李旻典()

陆军军医大学第一附属医院心血管内科,代谢生物钟与心血管病中心,重庆 400038

收稿日期:2022-07-15 修回日期:2022-09-02 出版日期:2022-10-20 发布日期:2022-09-13
通讯作者: 李旻典 E-mail:zengf719@163.com;mindianli@tmmu.edu.cn
作者简介:曾帆,硕士,研究实习员,研究方向：代谢生物钟与心血管疾病。E-mail: zengf719@163.com
基金资助:
国家自然科学基金青年科学基金项目(81900776)

Targeting leptin-positive adipocytes by expressing the Cre recombinase transgene under the endogenous leptin gene

Fan Zeng(), Lan Wang, Xiaoqin Wan, Rongfeng Huang, Zhihui Zhang, Min-Dian Li()

Department of Cardiovascular Medicine, Center for Circadian Metabolism and Cardiovascular Disease, Southwest Hospital, Army Medical University, Chongqing 400038, China

Received:2022-07-15 Revised:2022-09-02 Online:2022-10-20 Published:2022-09-13
Contact: Li Min-Dian E-mail:zengf719@163.com;mindianli@tmmu.edu.cn
Supported by:
the National Natural Science Foundation of China(81900776)

摘要/Abstract

摘要：

脂肪组织是发挥能量储存与内分泌功能的重要代谢组织。脂肪细胞的发育、稳态与病理生理功能是重要的研究方向。空间转录组学研究表明脂肪细胞至少存在3种不同亚型,其标记基因包括瘦素(leptin, LEP)、脂联素 (adiponectin, ADIPOQ)、围脂滴蛋白(perilipin-1/4, PLIN)和血清淀粉样蛋白(serum amyloid A, SAA)等。瘦素基因与脂联素基因分别标记了不同亚型的脂肪细胞。目前通常用脂联素基因Adipoq-Cre工具小鼠研究成熟脂肪细胞的生理功能,但缺乏瘦素基因启动的Cre工具小鼠以追踪LEP⁺亚型脂肪细胞的发育与稳态。本研究通过CRISPR-Cas9技术编辑小鼠Lep基因,形成Lep-P2A-Cre融合基因,其产物通过P2A肽的自剪切作用,解离为LEP与CRE蛋白,从而实现内源Lep基因启动的Cre表达(Lep-Cre)。本研究通过tdTomato示踪工具,分析Lep-Cre在不同脂肪组织与非脂肪组织的活性,结果发现Lep-Cre在白色脂肪和米色脂肪组织中活性最强,其次是棕色脂肪,在下丘脑核团不表达,但在多个外周器官存在不同程度的活性。本研究构建了瘦素基因启动的Lep-Cre工具小鼠,为深入研究LEP⁺亚型脂肪细胞的发育与功能提供新工具。

关键词: 瘦素, 脂肪细胞, Cre-loxP, 脂肪组织

Abstract:

Adipose tissue plays an important role in metabolic physiology through energy storage and endocrine functions. Spatial transcriptomics is revealing the complexity of cell types and their interaction in the adipose tissue with regards to development, homeostasis and disease. Emerging evidence suggests the existence of different subtypes of mature adipocytes that may have distinct functions, the markers of which include leptin (LEP), adiponectin (ADIPOQ), perilipin-1/4 (PLIN), and serum amyloid A (SAA), marking different adipocyte subtypes. Currently, Adipoq-Cre is widely used to study adipocyte biology, however, there is no Cre line that specifically targets LEP⁺adipocytes. Here, we report the construction and validation of a Lep-Cre mouse line, which has the endogenous Lep gene edited by the CRISPR-Cas9 technology to generate the Lep-peptide 2A (P2A)-Cre fusion gene. P2A induces an auto-hydrolysis of the fusion protein, leading to expression of the Cre recombinase by the Lep gene activity. The activity of Lep-Cre in different depots of adipose tissues and non-adipose tissues was visualized by the immunofluorescence microscopy in the Lep-Cre Rosa26-loxP-Stop-loxP-tdTomato mice. We showed that Lep-Cre marked white/beige adipose depots extensively, followed by brown adipose depots. Leaky activity was observed in varying degrees among peripheral organs but not in the paraventricular nucleus of the hypothalamus. In summary, we have constructed a new adipocyte-targeting Cre mouse line that would be useful to study the development and physiology of LEP⁺ adipocytes.

Key words: leptin, adipocyte, Cre-loxP, adipose tissue

曾帆, 王澜, 万小勤, 黄荣凤, 张志辉, 李旻典. 瘦素基因启动的新型脂肪细胞表达Cre工具小鼠的构建[J]. 遗传, 2022, 44(10): 950-957.

Fan Zeng, Lan Wang, Xiaoqin Wan, Rongfeng Huang, Zhihui Zhang, Min-Dian Li. Targeting leptin-positive adipocytes by expressing the Cre recombinase transgene under the endogenous leptin gene[J]. Hereditas(Beijing), 2022, 44(10): 950-957.

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基因型	引物序列	WT	Tg
Lep-Cre	F1:5′-CATATTGGCAGAACGAAAACGC-3′	NA	413 bp
Lep-Cre	R1:5′-CCTGTTTCACTATCCAGGTTACGG-3′
LSL-tdTomato	F2:5′-AAGGGAGCTGCAGTGGAGTA-3′	300 bp
	R2:5′-CCGAAAATCTGTGGGAAGTC-3′
	F3:5′-GGCATTAAAGCAGCGTATCC-3′		200 bp
	R3:5′-CTGTTCCTGTACGGCATGG-3′

瘦素基因启动的新型脂肪细胞表达Cre工具小鼠的构建

Targeting leptin-positive adipocytes by expressing the Cre recombinase transgene under the endogenous leptin gene

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