瘦素基因启动的新型脂肪细胞表达Cre工具小鼠的构建
收稿日期: 2022-07-15
修回日期: 2022-09-02
网络出版日期: 2022-09-13
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
Received date: 2022-07-15
Revised date: 2022-09-02
Online published: 2022-09-13
Supported by
the National Natural Science Foundation of China(81900776)
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。
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 . DOI: 10.16288/j.yczz.22-236
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
| [1] | Cypess AM. Reassessing human adipose tissue. N Engl J Med, 2022, 386(8): 768-779. |
| [2] | Zhang F, Hao GY, Shao ML, Nham K, An Y, Wang Q, Zhu Y, Kusminski CM, Hassan G, Gupta RK, Zhai QW, Sun XK, Scherer PE, Oz OK. An adipose tissue atlas: an image-guided identification of human-like BAT and beige depots in rodents. Cell Metab, 2018, 27(1): 252-262.e3. |
| [3] | Zhang ZH, Xin HR, Li MD. Circadian rhythm of lipid metabolism in health and disease. Small Methods, 2020, 4(7): 1900601. |
| [4] | Cohen P, Kajimura S. The cellular and functional complexity of thermogenic fat. Nat Rev Mol Cell Biol, 2021, 22(6): 393-409. |
| [5] | Sakers A, De Siqueira MK, Seale P, Villanueva CJ. Adipose- tissue plasticity in health and disease. Cell, 2022, 185(3): 419-446. |
| [6] | Lee KY, Luong Q, Sharma R, Dreyfuss JM, Ussar S, Kahn CR. Developmental and functional heterogeneity of white adipocytes within a single fat depot. EMBO J, 2019, 38(3): e99291. |
| [7] | Bäckdahl J, Franzén L, Massier L, Li Q, Jalkanen J, Gao H, Andersson A, Bhalla N, Thorell A, Rydén M, Ståhl PL, Mejhert N. Spatial mapping reveals human adipocyte subpopulations with distinct sensitivities to insulin. Cell Metab, 2021, 33(9): 1869-1882.e6. |
| [8] | Sárvári AK, Van Hauwaert EL, Markussen LK, Gammelmark E, Marcher AB, Ebbesen MF, Nielsen R, Brewer JR, Madsen JGS, Mandrup S. Plasticity of epididymal adipose tissue in response to diet-Induced obesity at single-nucleus resolution. Cell Metab, 2021, 33(2): 437-453.e5. |
| [9] | Min SY, Desai A, Yang Z, Sharma A, DeSouza T, Genga RMJ, Kucukural A, Lifshitz LM, Nielsen S, Scheele C, Maehr R, Garber M, Corvera S. Diverse repertoire of human adipocyte subtypes develops from transcriptionally distinct mesenchymal progenitor cells. Proc Natl Acad Sci USA, 2019, 116(36): 17970-17979. |
| [10] | Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng HK. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci, 2010, 13(1): 133-140. |
| [11] | Cancello R, Zingaretti MC, Sarzani R, Ricquier D, Cinti S. Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue. Endocrinology, 1998, 139(11): 4747-4750. |
| [12] | Charles KN, Li MD, Engin F, Arruda AP, Inouye K, Hotamisligil GS. Uncoupling of metabolic health from longevity through genetic alteration of adipose tissue lipid-binding proteins. Cell Rep, 2017, 21(2): 393-402. |
| [13] | Barlow C, Schroeder M, Lekstrom-Himes J, Kylefjord H, Deng CX, Wynshaw-Boris A, Spiegelman BM, Xanthopoulos KG. Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments. Nucleic Acids Res, 1997, 25(12): 2543-2545. |
| [14] | Shan TZ, Liu WY, Kuang SH. Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues. FASEB J, 2013, 27(1): 277-287. |
| [15] | Lee KY, Russell SJ, Ussar S, Boucher J, Vernochet C, Mori MA, Smyth G, Rourk M, Cederquist C, Rosen ED, Kahn BB, Kahn CR. Lessons on conditional gene targeting in mouse adipose tissue. Diabetes, 2013, 62(3): 864-874. |
| [16] | Straub LG, Scherer PE. Metabolic messengers: adiponectin. Nat Metab, 2019, 1(3): 334-339. |
| [17] | Wong AM, Patel TP, Altman EK, Tugarinov N, Trivellin G, Yanovski JA. Characterization of the adiponectin promoter + Cre recombinase insertion in the Tg(Adipoq-cre)1Evdr mouse by targeted locus amplification and droplet digital PCR. Adipocyte, 2021, 10(1): 21-27. |
| [18] | Pereira S, Cline DL, Glavas MM, Covey SD, Kieffer TJ. Tissue-specific effects of leptin on glucose and lipid metabolism. Endocr Rev, 2021, 42(1): 1-28. |
| [19] | Emont MP, Jacobs C, Essene AL, Pant D, Tenen D, Colleluori G, Di Vincenzo A, Jørgensen AM, Dashti H, Stefek A, McGonagle E, Strobel S, Laber S, Agrawal S, Westcott GP, Kar A, Veregge ML, Gulko A, Srinivasan H, Kramer Z, De Filippis E, Merkel E, Ducie J, Boyd CG, Gourash W, Courcoulas A, Lin SJ, Lee BT, Morris D, Tobias A, Khera AV, Claussnitzer M, Pers TH, Giordano A, Ashenberg O, Regev A, Tsai LT, Rosen ED. A single-cell atlas of human and mouse white adipose tissue. Nature, 2022, 603(7903): 926-933. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
