条件性RNAi敲低技术降低FGFR2的表达对小鼠软骨发育的影响
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摘要
基因敲除技术是研究基因功能的一种重要手段。基因敲除技术是80年代末以来发展起来的一种新型分子生物学技术，是通过一定的途径使机体特定的基因失活或缺失的技术。通常意义上的基因敲除主要是用DNA同源重组原理，用设计的基因片段代替靶基因片段，从而达到基因敲除的目的。随着基因敲除技术的发展，除了同源重组外，新的原理和技术也逐渐在被应用，如诱导性基因敲除技术、条件性基因敲除技术、利用随机插入片段进行基因敲除技术、反义核酸技术。
近年来，一些新技术也用于研究基因功能，RNA干扰就是其中的一种。RNAi技术是使基因表达降低，通过比较前后基因型的变化，能很方便的鉴定出基因的功能。本实验是用条件性RNAi基因敲低技术来研究小鼠软骨细胞中FGFR2对小鼠软骨发育的作用。
首先，鉴定出条件性RNAi敲低FGFR2小鼠FGFR2-RNAi和在软骨细胞特异表达Cre重组蛋白的转基因小鼠Coll2a-Cre，然后让FGFR2-RNAi小鼠和在软骨细胞特异表达Cre重组蛋白的转基因小鼠Coll2a-Cre交配，经过对后代小鼠的基因型鉴定，我们得到双转基因小鼠FGFR2RNAi；Coll2a-Cre，我们观察了雌性双转基因小鼠FGFR2RNAi；Coll2a-Cre和同窝雌性对照体重和时间的关系以及雄性双转基因小鼠FGFR2RNAi；Coll2a-Cre和同窝雄性对照体重和时间的关系，经过统计学分析，发现双转基因小鼠FGFR2RNAi；Coll2a-Cre要比同窝对照小鼠的体重要轻；并进一步分别观察了雌、雄性双转基因小鼠FGFR2RNAi；Coll2a-Cre与同窝对照小鼠的生长板宽度，并进行了统计学分析，发现双转基因小鼠FGFR2RNAi；Coll2a-Cre要比同窝对照小鼠生长板的宽度要小；经过软骨细胞的体外培养，并进行FGFR2的RT-PCR分析，我们发现双转基因小鼠FGFR2RNAi；Coll2a-Cre软骨细胞中FGFR2mRNA相对同窝对照表达降低。上述结果提示可用条件性RNAi敲低技术降低FGFR2在软骨中的表达来观察软骨的发育情况。
Gene knockout is an important method to investigate the role of gene.Geneknockout technology has already become a new molecular biology technology by theend of 1980's.As usually, Gene knockou is based on the, which use the designedgene fragment to take the place of target gene fragment to achieve the goal of geneknockout.With the development of gene knockout,new principle and technology isincreasingly used, such as,inducible gene knockout, conditional gene knockout,random insertion fragment for gene knockout, antisense nuclear acid technology.In recent years,some new technology is used in research on gene role,RNAiknockdown technology, as one of them,decreases gene expression and identifieseasily gene role by comparison of genetype. In this article, we investigate the impactof FGFR2 in mouse cartilage on the development of cartilage with conditional RNAiknockdown technology.
Firstly, we identify the conditional FGFR2 knockdown FGFR2-RNAi mouseand Coll2a-Cre mouse expressing recombination protein Cre in cartilage.Then,wemake FGFR2-RNAi mouse mate with Coll2a-Cre mouse to produce offspringdouble transgenic mouse FGFR2RNAi; Coll2a-Cre identified by genetype, we havethe reaserch on the relationship of weight and time for female double transgenicmouse and female control and find that weight of female double transgenic mouse isless than female control in the same time after statistical analysis.For the maledouble transgenic mouse and male control, we have got the same result above.wefurther compare the width of female double transgenic and male double transgenic tocontrol respectively and find that the width of double transgenic mouse is less thancontrol in the same time after statistic analysis. We culture cartilage cell in vivo andanalyze RT-PCR of FGFR2 and find that the content of FGFR2mRNA in cartilage ofFGFR2RNAi; Coll2a-Cre mice is lower than that of controls.Therefore,all the resultsabove release that we can observe the development of cartilage with conditionalRNAi knockdown decreasing FGFR2 expression.
近年来，一些新技术也用于研究基因功能，RNA干扰就是其中的一种。RNAi技术是使基因表达降低，通过比较前后基因型的变化，能很方便的鉴定出基因的功能。本实验是用条件性RNAi基因敲低技术来研究小鼠软骨细胞中FGFR2对小鼠软骨发育的作用。
首先，鉴定出条件性RNAi敲低FGFR2小鼠FGFR2-RNAi和在软骨细胞特异表达Cre重组蛋白的转基因小鼠Coll2a-Cre，然后让FGFR2-RNAi小鼠和在软骨细胞特异表达Cre重组蛋白的转基因小鼠Coll2a-Cre交配，经过对后代小鼠的基因型鉴定，我们得到双转基因小鼠FGFR2RNAi；Coll2a-Cre，我们观察了雌性双转基因小鼠FGFR2RNAi；Coll2a-Cre和同窝雌性对照体重和时间的关系以及雄性双转基因小鼠FGFR2RNAi；Coll2a-Cre和同窝雄性对照体重和时间的关系，经过统计学分析，发现双转基因小鼠FGFR2RNAi；Coll2a-Cre要比同窝对照小鼠的体重要轻；并进一步分别观察了雌、雄性双转基因小鼠FGFR2RNAi；Coll2a-Cre与同窝对照小鼠的生长板宽度，并进行了统计学分析，发现双转基因小鼠FGFR2RNAi；Coll2a-Cre要比同窝对照小鼠生长板的宽度要小；经过软骨细胞的体外培养，并进行FGFR2的RT-PCR分析，我们发现双转基因小鼠FGFR2RNAi；Coll2a-Cre软骨细胞中FGFR2mRNA相对同窝对照表达降低。上述结果提示可用条件性RNAi敲低技术降低FGFR2在软骨中的表达来观察软骨的发育情况。
Gene knockout is an important method to investigate the role of gene.Geneknockout technology has already become a new molecular biology technology by theend of 1980's.As usually, Gene knockou is based on the, which use the designedgene fragment to take the place of target gene fragment to achieve the goal of geneknockout.With the development of gene knockout,new principle and technology isincreasingly used, such as,inducible gene knockout, conditional gene knockout,random insertion fragment for gene knockout, antisense nuclear acid technology.In recent years,some new technology is used in research on gene role,RNAiknockdown technology, as one of them,decreases gene expression and identifieseasily gene role by comparison of genetype. In this article, we investigate the impactof FGFR2 in mouse cartilage on the development of cartilage with conditional RNAiknockdown technology.
Firstly, we identify the conditional FGFR2 knockdown FGFR2-RNAi mouseand Coll2a-Cre mouse expressing recombination protein Cre in cartilage.Then,wemake FGFR2-RNAi mouse mate with Coll2a-Cre mouse to produce offspringdouble transgenic mouse FGFR2RNAi; Coll2a-Cre identified by genetype, we havethe reaserch on the relationship of weight and time for female double transgenicmouse and female control and find that weight of female double transgenic mouse isless than female control in the same time after statistical analysis.For the maledouble transgenic mouse and male control, we have got the same result above.wefurther compare the width of female double transgenic and male double transgenic tocontrol respectively and find that the width of double transgenic mouse is less thancontrol in the same time after statistic analysis. We culture cartilage cell in vivo andanalyze RT-PCR of FGFR2 and find that the content of FGFR2mRNA in cartilage ofFGFR2RNAi; Coll2a-Cre mice is lower than that of controls.Therefore,all the resultsabove release that we can observe the development of cartilage with conditionalRNAi knockdown decreasing FGFR2 expression.
引文
[1] Maximilian Muenke, Ute Schell. Fibroblast-growth-factor receptor mutationsin human skeletal disordersJ. TIG 1995,11 (8):308
[2] Alexander N Plothikov, Joseph Schlessinger,Stevan R Hubbard,et al. Structural basis for FGF receptor dimerization and activationJ. Cell,1999,98:641.
[3] 王丽颖等．成纤维生长因子受体研究进展J．国外医学免疫学分册，1998，21(4)：175．
[4] Anne Hanneken. Structural Characterization of the circulatingouble FGF receptors reveals multiple isoforms generated by secretion and ectodomain sheddingJ. FEBS Letters,2001,489: 176.
[5] Coumoul X, Deng CX. Roles of FGF Receptors in Mammalian Development and Congenital Diseases. Birth Defects Res, 2003,69(4):286-304.
[6] Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev,2002, 16(12): 1446-65.
[7] Prawitt D, Brixel L, Spangenberg C et al. RNAi knock-down mice: an emerging technology for post-genomic functional genetics. Cytogenet Genome Res, 2004, 105(2-4):412-21.
[8] Anne -Lise Delezoide, Catherline Benoist-Lasselin,Lautence Legeai-Mallet,et al. Spatio-temporal expression of FGFR1,2 and 3 genes during human embryo-fetal ossification J. Mecha- nisms of Development, 1998, 77:19.
[9] Eriebacher A, Filvaroff E H, Gitelman S E, et al. Toward a Molecular Understanding of Skeletal Development J. Cell, 1995,80:371.
[10] Webster M K, Dono ghue DJ. FGFR activation in skeletal dis orders: too much of a good thing J. TIG MAY,1997,13, (5):178.
[11] Nabel G; Yan g Z Y, Plautz G, et al. Recombinant fibroblast growthfactor-1 promotesintimal hyperplasia and angiogenesis in arteries in vivo J. Nature, 1993,362:844
[12] Yana gisawa-Miwa A, Uchidz Y, Nakamora F, et al. Salvage of infracted myocardium by angiogenic action of basic fibroblast growth factor J Science ,1992 ,257:140121403.
[2] Alexander N Plothikov, Joseph Schlessinger,Stevan R Hubbard,et al. Structural basis for FGF receptor dimerization and activationJ. Cell,1999,98:641.
[3] 王丽颖等．成纤维生长因子受体研究进展J．国外医学免疫学分册，1998，21(4)：175．
[4] Anne Hanneken. Structural Characterization of the circulatingouble FGF receptors reveals multiple isoforms generated by secretion and ectodomain sheddingJ. FEBS Letters,2001,489: 176.
[5] Coumoul X, Deng CX. Roles of FGF Receptors in Mammalian Development and Congenital Diseases. Birth Defects Res, 2003,69(4):286-304.
[6] Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev,2002, 16(12): 1446-65.
[7] Prawitt D, Brixel L, Spangenberg C et al. RNAi knock-down mice: an emerging technology for post-genomic functional genetics. Cytogenet Genome Res, 2004, 105(2-4):412-21.
[8] Anne -Lise Delezoide, Catherline Benoist-Lasselin,Lautence Legeai-Mallet,et al. Spatio-temporal expression of FGFR1,2 and 3 genes during human embryo-fetal ossification J. Mecha- nisms of Development, 1998, 77:19.
[9] Eriebacher A, Filvaroff E H, Gitelman S E, et al. Toward a Molecular Understanding of Skeletal Development J. Cell, 1995,80:371.
[10] Webster M K, Dono ghue DJ. FGFR activation in skeletal dis orders: too much of a good thing J. TIG MAY,1997,13, (5):178.
[11] Nabel G; Yan g Z Y, Plautz G, et al. Recombinant fibroblast growthfactor-1 promotesintimal hyperplasia and angiogenesis in arteries in vivo J. Nature, 1993,362:844
[12] Yana gisawa-Miwa A, Uchidz Y, Nakamora F, et al. Salvage of infracted myocardium by angiogenic action of basic fibroblast growth factor J Science ,1992 ,257:140121403.