利用CRISPR/Cas9系统敲除绵羊皮肤成纤维细胞Wnt2基因的研究
|
摘要
为了研究无翅基因相关整合位点2(Wnt2)在绵羊毛囊发育中的作用,通过CRISPR/Cas9系统介导的基因编辑技术在绵羊原代皮肤成纤维细胞中敲除Wnt2基因,经T7E1酶切试验进行敲除结果验证,实时荧光定量PCR分析Wnt2、E-钙黏蛋白(CDH1)、半光天冬氨酸酶3(Caspase3)和成纤维细胞生长因子5(FGF5)在敲除后的表达量变化。结果显示:绵羊原代皮肤成纤维细胞中Wnt2基因敲除成功,编辑效率约14.35%;Wnt2相对表达量极显著下调(P<0.01),CDH1相对表达量极显著上调(P<0.01),Caspase3及FGF5表达上调显著(P<0.05)。结果表明:Wnt2基因对毛囊的生长发育具有促进作用。
The CRISPR/Cas9 system was used in the present experiment to knockout Wnt2 in the primary cell of sheep skin fibroblast.T7 E1 enzyme digestion was employed to verify the knockout result. And RT-PCR was performed to analyze differences in the expression of Wnt2,CDH1,caspase3 and FGF5 after knockout. The results show that the Wnt2 knockout was successful with the editing efficiency being14. 35%. The expression of Wnt2 was down-regulated very significantly(P<0. 01),and the expression of CDH1 was up-regulated very significantly(P<0. 01). The expressions of FGF5 and caspase3 were significantly up-regulated(P< 0. 05). To conclude,Wnt2 promotes the growth and development of hair follicles.
The CRISPR/Cas9 system was used in the present experiment to knockout Wnt2 in the primary cell of sheep skin fibroblast.T7 E1 enzyme digestion was employed to verify the knockout result. And RT-PCR was performed to analyze differences in the expression of Wnt2,CDH1,caspase3 and FGF5 after knockout. The results show that the Wnt2 knockout was successful with the editing efficiency being14. 35%. The expression of Wnt2 was down-regulated very significantly(P<0. 01),and the expression of CDH1 was up-regulated very significantly(P<0. 01). The expressions of FGF5 and caspase3 were significantly up-regulated(P< 0. 05). To conclude,Wnt2 promotes the growth and development of hair follicles.
引文
[1]Lee A,Bae S,Lee S H,et al.Hair follicles serve as local reservoirs of skin mast cell precursors[J].Journal of Veterinary Medical Science,2016,78(12):1811-1818.
[2]Roy E,Neufeld Z,Cerone L,et al.Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling[J].The EMBO Journal,2016,35(24):2628-2630.
[3]Andl T,Rebby S T,Gaddapara T,et al.WNT signals are required for the initiation of hair follicle development[J].Developmental Cell,2002,2(5):643-653.
[4]Katoh M.Pancreatic neuroendocrine tumor cancer stem cells:potential novel therapeutic targets?[J].Translational Cancer Research,2016,5(6):860-870.
[5]Yue Y J,Guo T T,Yuan C,et al.Integrated analysis of the roles of long noncoding RNA and coding RNA expression in sheep(Ovis aries)skin during initiation of secondary hair follicle[J].Plo S One,2016,11(6).
[6]Gaj T,Gersbach C A,Barbas C F.ZFN,TALEN,and CRISPR/Cas-based methods for genome engineering[J].Trends in Biotechnology,2013,31(7):397-405.
[7]Hsu P D,Lander E,Zhang F,et al.Development and Applications of CRISPR-Cas9 for Genome Engineering[J].Cell,2014,157(6):1262-1278.
[8]Kwan V,Unda B K,Singh K K.Wnt signaling networks in autism spectrum disorder and intellectual disability[J].Journal of Neurodevelopmental Disorders,2016,(8):45.
[9]Chamorro M N,Schwartz D R,Vonica A,et al.FGF-20 and DKK1are transcriptional targets of beta-catenin and FGF-20 is implicated in cancer and development[J].The EMBO Journal,2005,24:73-84.
[10]Masuko K,Masaru K.WNT Signaling Pathway and Stem Cell Signaling Network[J].Clinical Cancer Research,2007,13(14):4042-4045.
[11]Yoshizawa Y,Wada K,Shiomi G,et al.A 1-bp deletion in Fgf5causes male-dominant long hair in the Syrian hamster[J].Mammalian Genome,2015,26:11-12.
[12]Bose S K,Meyer K,Di Bisceglle A M,et al.Hepatitis C virus induces epithelial-mesenchymal transition in primary human hepatocytes[J].Journal of Virology,2012,86:13621-13628.
[13]Fuchsberger T,Martinez-Bellver S,Giraldo E,et al.A beta induces excitotoxicity mediated by APC/C-Cdh1 depletion that can be prevented by glutaminase inhibition promoting neuronal survival[J].Scientific Reports,2016,(6).
[14]You L,He B,Xu Z D,et al.An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth[J].Cancer Research,2004,64(15):5385-5389.
[2]Roy E,Neufeld Z,Cerone L,et al.Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling[J].The EMBO Journal,2016,35(24):2628-2630.
[3]Andl T,Rebby S T,Gaddapara T,et al.WNT signals are required for the initiation of hair follicle development[J].Developmental Cell,2002,2(5):643-653.
[4]Katoh M.Pancreatic neuroendocrine tumor cancer stem cells:potential novel therapeutic targets?[J].Translational Cancer Research,2016,5(6):860-870.
[5]Yue Y J,Guo T T,Yuan C,et al.Integrated analysis of the roles of long noncoding RNA and coding RNA expression in sheep(Ovis aries)skin during initiation of secondary hair follicle[J].Plo S One,2016,11(6).
[6]Gaj T,Gersbach C A,Barbas C F.ZFN,TALEN,and CRISPR/Cas-based methods for genome engineering[J].Trends in Biotechnology,2013,31(7):397-405.
[7]Hsu P D,Lander E,Zhang F,et al.Development and Applications of CRISPR-Cas9 for Genome Engineering[J].Cell,2014,157(6):1262-1278.
[8]Kwan V,Unda B K,Singh K K.Wnt signaling networks in autism spectrum disorder and intellectual disability[J].Journal of Neurodevelopmental Disorders,2016,(8):45.
[9]Chamorro M N,Schwartz D R,Vonica A,et al.FGF-20 and DKK1are transcriptional targets of beta-catenin and FGF-20 is implicated in cancer and development[J].The EMBO Journal,2005,24:73-84.
[10]Masuko K,Masaru K.WNT Signaling Pathway and Stem Cell Signaling Network[J].Clinical Cancer Research,2007,13(14):4042-4045.
[11]Yoshizawa Y,Wada K,Shiomi G,et al.A 1-bp deletion in Fgf5causes male-dominant long hair in the Syrian hamster[J].Mammalian Genome,2015,26:11-12.
[12]Bose S K,Meyer K,Di Bisceglle A M,et al.Hepatitis C virus induces epithelial-mesenchymal transition in primary human hepatocytes[J].Journal of Virology,2012,86:13621-13628.
[13]Fuchsberger T,Martinez-Bellver S,Giraldo E,et al.A beta induces excitotoxicity mediated by APC/C-Cdh1 depletion that can be prevented by glutaminase inhibition promoting neuronal survival[J].Scientific Reports,2016,(6).
[14]You L,He B,Xu Z D,et al.An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth[J].Cancer Research,2004,64(15):5385-5389.