毛囊特异表达载体pUHS-FGF18的构建及转基因小鼠的制备

Abstract
Figure/Table
References
Related Citation (15)

Download: PDF (1615 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Fibroblast growth factor 18 (Fgf18) is a secreted signaling molecule and involved in regulating hair growth cycle of mouse (Mus musculus) and cashmere goat (Capra hircus). Primers attached with XbaⅠ and SacⅠ restriction enzymes were designed to clone Fgf18 CDS sequence, and then the PCR product was ligated to the downstream of ultra-high sulfur keratin (UHS) promoter. For application in somatic cell nuclear transfer (SCNT), enhanced green fluorescent protein gene (EGFP) and neomycin gene (Neo) promoted by cytomegalovirus (CMV) promoter were added into the recombinant plasmid. Four restriction enzymes (SacⅠ, BglⅡ, SphⅠ and EcoT14Ⅰ) digestion results showed that the follicle-specific expression vector of Fgf18 gene was successfully constructed, named as pUHS-Fgf18. Three positive transgenic mice were obtained by using pronuclear microinjection, and the integration efficiency was 5.8%. RT-PCR and Western blot results showed that the expression levels of Fgf18 were significantly increased in the skin of transgenic mice, compared to controls (P＜0.05). In order to verify the tissue-specific expression, the expression levels of Fgf18 mRNA were also detected in transgenic mice skin, liver, kidney, heart, testis, skeletal muscle, spleen, brain, lung, stomach and small intestine tissues by using RT-PCR. The results showed that the expression levels of Fgf18 mRNA were significantly increased in transgenic mouse skin (P＜0.05) and there were no significant difference in other tissues between transgenic mouse and controls (P＞0.05). Above mentioned research indicated that pUSH-Fgf18 vector can specifically express Fgf18 in skin. Then RT-PCR was used to detect the copy number of Fgf18 in transgenic mice. The result showed that they were ranging from 8~210, and no copy was detected in controls. This study successfully established a transgenic mouse model, which can serve as a favorable tool to study the function and mechanism of Fgf18 in hair growth cycle, and will provide a theoretical basis to produce Fgf18 gene modified cashmere goats.

Key words： Fibroblast growth factor 18(Fgf18) Hair follicle Tissue specificity Ultra-high sulfur keratin(UHS) Transgenic mice

Received: 15 February 2016 Published: 01 July 2016

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	LI Xin-Xin
	YU Xu-Dong
	LI Dong-Fang
	WEI Zhao-Yang
	YIN Feng-Xia
	YANG Lei
	LI Guang-Peng

Cite this article:

LI Xin-Xin,YU Xu-Dong,LI Dong-Fang, et al. Construction of the Follicle-specific Expression Vector for Fgf18 and Production of Transgenic Mouse (Mus musculus)[J]. , 2016, 24(8): 1165-1172.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2016/V24/I8/1165

Blessing M, Bhagwat MS, Lyatskaya Y, et al. 2012. Kilovoltage beam model for flat panel imaging system with bow-tie filter for scatter prediction and correction[J]. Phys Med, 28(2): 134-43.
Copeland NG, Jenkins NA, and Court DL 2001. Recombineering: a powerful new tool for mouse functional genomics[J]. Nat Rev Genet, 2(10): 769-79.
Ding Q, and Gan L 2012. Conditional control of gene expression in the mouse retina[J]. Methods Mol Biol, 884(3-15.
Giraldo P, and Montoliu L 2001. Size matters: use of YACs, BACs and PACs in transgenic animals[J]. Transgenic Res, 10(2): 83-103.
Grieshammer U, Lewandoski M, Prevette D, et al. 1998. Muscle-specific cell ablation conditional upon Cre-mediated DNA recombination in transgenic mice leads to massive spinal and cranial motoneuron loss[J]. Dev Biol, 197(2): 234-47.
Hansen LA, Alexander N, Hogan ME, et al. 1997. Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development[J]. Am J Pathol, 150(6): 1959-75.
Haque T, Nakada S, and Hamdy RC 2007. A review of FGF18: Its expression, signaling pathways and possible functions during embryogenesis and post-natal development[J]. Histol Histopathol, 22(1): 97-105.
Hibino T, and Nishiyama T 2004. Role of TGF-β2 in the human hair cycle[J]. Journal of Dermatological Science, 35(1): 9-18.
Imamura T 2014. Physiological functions and underlying mechanisms of fibroblast growth factor (FGF) family members: recent findings and implications for their pharmacological application[J]. Biol Pharm Bull, 37(7): 1081-9.
Kashiwagi M, Kuroki T, and Huh N 1997. Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin[J]. Dev Biol, 189(1): 22-32.
Kawano M, Komi-Kuramochi A, Asada M, et al. 2005. Comprehensive analysis of FGF and FGFR expression in skin: FGF18 is highly expressed in hair follicles and capable of inducing anagen from telogen stage hair follicles[J]. J Invest Dermatol, 124(5): 877-85.
Kimura-Ueki M, Oda Y, Oki J, et al. 2012. Hair Cycle Resting Phase Is Regulated by Cyclic Epithelial FGF18 Signaling[J]. Journal of Investigative Dermatology, 132(5): 1338-1345.
Kozlowska U, Blume-Peytavi U, Kodelja V, et al. 1998. Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle[J]. Arch Dermatol Res, 290(12): 661-8.
Lee YR, Yamazaki M, Mitsui S, et al. 2001. Hepatocyte growth factor (HGF) activator expressed in hair follicles is involved in in vitro HGF-dependent hair follicle elongation[J]. J Dermatol Sci, 25(2): 156-63.
Lindner G, Menrad A, Gherardi E, et al. 2000. Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle morphogenesis and cycling[J]. Faseb J, 14(2): 319-32.
Ohbayashi N, Hoshikawa M, Kimura S, et al. 1998. Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF-18[J]. J Biol Chem, 273(29): 18161-4.
Paus R, Foitzik K, Welker P, et al. 1997. Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling[J]. J Invest Dermatol, 109(4): 518-26.
Paus R, Muller-Rover S, and Botchkarev VA 1999. Chronobiology of the hair follicle: hunting the " hair cycle clock"[J]. J Investig Dermatol Symp Proc, 4(3): 338-45.
Pittelkow MR, and Shipley GD 1989. Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements[J]. J Cell Physiol, 140(3): 565-76.
Rosenquist TA, and Martin GR 1996. Fibroblast growth factor signalling in the hair growth cycle: expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle[J]. Dev Dyn, 205(4): 379-86.
Su HY, Hickford JG, The PH, et al. 1999. Increased vibrissa growth in transgenic mice expressing insulin-like growth factor 1[J]. J Invest Dermatol, 112(2): 245-8.
Suzuki S, Kato T, Takimoto H, et al. 1998. Localization of rat FGF-5 protein in skin macrophage-like cells and FGF-5S protein in hair follicle: possible involvement of two Fgf-5 gene products in hair growth cycle regulation[J]. J Invest Dermatol, 111(6): 963-72.
Suzuki S, Ota Y, Ozawa K, et al. 2000. Dual-mode regulation of hair growth cycle by two Fgf-5 gene products[J]. J Invest Dermatol, 114(3): 456-63.
Tomita Y, Akiyama M, and Shimizu H 2006. PDGF isoforms induce and maintain anagen phase of murine hair follicles[J]. J Dermatol Sci, 43(2): 105-15.
Trentin-Sonoda M, da Silva RC, Kmit FV, et al. 2015. Knockout of Toll-Like Receptors 2 and 4 Prevents Renal Ischemia-Reperfusion-Induced Cardiac Hypertrophy in Mice[J]. PLoS One, 10(10).
Vandenabeele SI, Daminet S, Van Ham L, et al. 2011. Immunohistochemical localization of fibroblast growth factor 18 in hair follicles of healthy beagle dogs[J]. Vet Dermatol, 22(5): 423-8.
Weger N 2005. IGF-I Signalling Controls the Hair Growth Cycle and the Differentiation of Hair Shafts[J]. The Society for Investigative Dermatology, 125(
Wood L, Mills M, Hatzenbuhler N, et al. 1991. Serine-rich ultra high sulfur protein gene expression in murine hair and skin during the hair cycle[J]. J Biol Chem., 266(6): 4024.
Yang L, Mao C, Teng Y, et al. 2005. Targeted disruption of Smad4 in mouse epidermis results in failure of hair follicle cycling and formation of skin tumors[J]. Cancer Res, 65(19): 8671-8.