qPCR array检测分析C57BL/6小鼠胚胎后肢发育基因表达谱
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摘要
目的:胚胎生育过程中因肢体发育异常造成的出生缺陷比率不低,其相关基因表达模式尚不明确。本实验通过建立实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究C57BL/6品系小鼠后肢发育相关基因的表达谱。方法:以同源异形盒基因家族(Hox)、Wnt5a、配对同源结构域基因(Pitx1)、成纤维生长因子(Fgf8)、音猬因子(Shh)等小鼠肢体发育相关的重要基因制作基因检测表达谱,以C57BL/6品系怀孕雌鼠为材料,取胚胎肢芽发育的四个关键时期(E10.5,E11.5,E12.5,E13.5)的胎鼠后肢,利用qPCR array方案检测表达谱中基因的相对表达水平差异。结果:通过已建立的qPCR array检测了C57BL/6品系小鼠胚胎后肢发育时期Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因的表达差异。以E10.5为对照,检测出在后肢发育时期基因呈三种表达模式,即Hoxb6、Hoxb8、Hoxc8、Hoxc9、Hoxc10、Hoxd9和Shh基因的表达水平呈上调;Hoxa11、Hoxa13、Hoxc12、Hoxc13、Hoxd13等基因表达出现下调;Hoxc9、Hoxc10、Hoxc11、Hoxd9、Hoxd12、Fgf8和Pitx1等基因的相对表达量呈先上调后下调的曲线表达模式,且有少部分基因在小鼠后肢发育时期表达水平无明显变化。结论:Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因在小鼠后肢发育时期表达,并且表达模式存在明显差异。
Objective: The rates of birth defects caused by abnormal limb development are not low in the process of embryo development, however, the expression patterns of the related genes are not clear. In this study, we established a real-time quantitative polymerasechain reaction array method to study the expression of genes related to hind limb development in C57 BL/6 mouse strains. Methods:Hox, Wnt5 a, Pitx1, Fgf8 and Shh were used to make the expression profiles of the genes related to hind limb development in mice. The hind limbs of the fetal C57 BL/6 mice were taken from the four critical stages(E10.5, E11.5, E12.5, E13.5) of embryonic limb buds development. We detected relative genetic expression levels by the qPCR array. Results: The expression levels of Hox, Wnt5 a, Pitx1, Fgf8 and Shh were detected during the development of the C57 BL/6 mouse strains hind limbs by the qPCR array. Using E10.5 as a control, we detected that the genes presented the three expression patterns during the development of the hind limbs. The expression levels of Hoxb6,Hoxb8, Hoxc8, Hoxc9, Hoxc10, Hoxd9 and Shh genes were up-regulated. And the expression levels of Hoxa11, Hoxa13, Hoxc12,Hoxc13 and Hoxd13 genes were down-regulated. The relative expression levels of Hoxc9, Hoxc10, Hoxc11, Hoxd9, Hoxd12, Fgf8 and Pitx1 genes were up-regulated and then down-regulated. And there were no significant changes in the expression levels of a small number of genes. Conclusions: Hox, Wnt5 a, Pitx1, Fgf8 and Shh genes were expressed in the development of mice hind limbs, and their expression patterns were significantly different.
Objective: The rates of birth defects caused by abnormal limb development are not low in the process of embryo development, however, the expression patterns of the related genes are not clear. In this study, we established a real-time quantitative polymerasechain reaction array method to study the expression of genes related to hind limb development in C57 BL/6 mouse strains. Methods:Hox, Wnt5 a, Pitx1, Fgf8 and Shh were used to make the expression profiles of the genes related to hind limb development in mice. The hind limbs of the fetal C57 BL/6 mice were taken from the four critical stages(E10.5, E11.5, E12.5, E13.5) of embryonic limb buds development. We detected relative genetic expression levels by the qPCR array. Results: The expression levels of Hox, Wnt5 a, Pitx1, Fgf8 and Shh were detected during the development of the C57 BL/6 mouse strains hind limbs by the qPCR array. Using E10.5 as a control, we detected that the genes presented the three expression patterns during the development of the hind limbs. The expression levels of Hoxb6,Hoxb8, Hoxc8, Hoxc9, Hoxc10, Hoxd9 and Shh genes were up-regulated. And the expression levels of Hoxa11, Hoxa13, Hoxc12,Hoxc13 and Hoxd13 genes were down-regulated. The relative expression levels of Hoxc9, Hoxc10, Hoxc11, Hoxd9, Hoxd12, Fgf8 and Pitx1 genes were up-regulated and then down-regulated. And there were no significant changes in the expression levels of a small number of genes. Conclusions: Hox, Wnt5 a, Pitx1, Fgf8 and Shh genes were expressed in the development of mice hind limbs, and their expression patterns were significantly different.
引文
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[18]杨媛媛.小鼠胚胎发育形态观察及CRB3在胚胎和成体中的表达[D].陕西:西北农林科技大学,2011Yang Yuan-yuan.Morphological observation on mouse embryo development and expression of CRB3 in mouse embryo and adult body[D].Shanxi:Northwest A&F University,2011
[19]N Wanek,K Muneoka,G Holler-Dinsmore,et al.A Staging System for Mouse Limb Development[J].The Journal of Experimental Zoology,1989,249:41-49
[20]Rulang Jiang,Yu Lan,Harry D.Chapman,et al.Defects in limb,craniofacial,and thymic development in Jagged2 mutant mice[J].Genes&Development,1998,12:1046-1057
[21]Franck Rapapport,Raya Khanin,Yupu Liang,et al.Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data[J].Genome Biololgy,2013,14:3158-3170
[22]Guillaume Andrey,Denis Duboule.Snap Shot:Hox Gene Regulation[J].Cell,2014,156:856
[2]Alan R.Rodrigues,Nayuta Yakushiji-Kaminatsui,Yuji Atsuta,et al.Integration of Shh and Fgf signaling in controlling Hox gene expression in cultured limb cells[J].PNAS,2017,114(12):3139-3144
[3]Daniel P Szeto,Concepcion Rodriguez-Esteban,Aimee K.Ryan,et al.Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development[J].Genes&Development,1999,13:484-494
[4]刘厚琴,王毅.WNT信号通路研究进展[J].社区医学杂志,2015,13(5):15-18Liu Hou-qin,Wang Yi.Advances in WNT signaling pathway[J].Journal of Community Medicine,2015,13(5):15-18
[5]Wang Yi-ping,Li Yi-ping,Christie Paulson,et al.Wnt and the Wnt signaling pathway in bone development and disease[J].NIH Public Access,2014,19:379-407
[6]刘凤松,胡钦亮,杜世新.Hox基因在马蹄内翻足病因中的研究进展[J].天津医药,2015,43(6):702-704Liu Feng-song,Hu Qin-liang,Du Shi-xin.Current research of role of Hox genes in pathogenesis of equinus deformity[J].Tianjin Med J,2015,43(6):702-704
[7]金淑清,浦予飞,裘莹.Hox基因的研究进展[J].癌症进展,2011,9(2):154-158Jin Shu-qing,Pu Shu-feng,Qiu Ying.The study advances of HOX gene[J].Oncology Progress,2011,9(2):154-158
[8]朱长保,徐福意,晁天柱,等.q PCR array检测高糖对胆固醇合成基因表达的影响[J].现代生物医学进展,2014,14(33):6420-6424Zhu Chang-bao,Xu Fu-yi,Chao Tian-zhu,et al.Effect of High Glucose on the Expression of Cholesterol Biosynthesis Gene by q PCR Array[J].Progress in Modern Biomedicine,2014,14(33):6420-6424
[9]Jensen KK,Previs SF,Zhu L,et al.Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification[J].Am J Physiol Endocrinol Metab,2012,302(2):209-217
[10]Eugeniu Nacu,Elena Gromberg,Catarina R.Oliveira,et al.FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration[J].Nature,2016,533:407-422
[11]Candace Rapchak,Neeraj Patel,John Hudson,et al.Conservation of Pitx1 expression during amphibian limb morphogenesis[J].Biochemistry and Cell Biology,2015,93(4):396-404
[12]Marco Osterwalder,Dario Speziale,Malak Shoukry,et al.HAND2Targets Define a Network of Transcriptional Regulators that Compartmentalize the Early Limb Bud Mesenchyme[J].Developmental Cell,2014,31(3):345-357
[13]Kyriel M.Pineault,Deneen M.Wellik.Hox Genes and Limb Musculoskeletal Development[J].Current Osteoporosis Reports,2014,12(4):420-427
[14]Yo-ichi Yamamoto-Shiraishi,Atsushi Kuroiwa.Wnt and BMP signaling cooperate with Hox in the control of Six2 expression in limb tendon precursor[J].Developmental Biology,2013,377(2):363-374
[15]Masaru Tamura,Masaki Hosoya,Motoi Fujita,et al.Overdosage of Hand2 causes limb and heart defects in the human chromosomal disorder partial trisomy distal 4q[J].Human Molecular Genetics,2013,22(12):2471-2481
[16]Malte Spielmann,Naseebullah Kakar,Naeimeh Tayebi,et al.Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice[J].Genome Research,2016,26(2):183-191
[17]V.Duboc,MP Logan.Regulation of limb bud initiation and limb-type morphology[J].Developmental dynamics,2011,240(5):1017-1027
[18]杨媛媛.小鼠胚胎发育形态观察及CRB3在胚胎和成体中的表达[D].陕西:西北农林科技大学,2011Yang Yuan-yuan.Morphological observation on mouse embryo development and expression of CRB3 in mouse embryo and adult body[D].Shanxi:Northwest A&F University,2011
[19]N Wanek,K Muneoka,G Holler-Dinsmore,et al.A Staging System for Mouse Limb Development[J].The Journal of Experimental Zoology,1989,249:41-49
[20]Rulang Jiang,Yu Lan,Harry D.Chapman,et al.Defects in limb,craniofacial,and thymic development in Jagged2 mutant mice[J].Genes&Development,1998,12:1046-1057
[21]Franck Rapapport,Raya Khanin,Yupu Liang,et al.Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data[J].Genome Biololgy,2013,14:3158-3170
[22]Guillaume Andrey,Denis Duboule.Snap Shot:Hox Gene Regulation[J].Cell,2014,156:856