目的:探讨siRNA抑制TGFβ3诱发腭裂的机制。方法:使用不同剂量(200、400、800pmol)经脂质体LipofectamineTM2000包裹的TGFβ3 StealthTMRNAi于交配后(days post coitum,dpc)12天做孕鼠宫腔注射,等体积生理盐水注射作为空白对照。14dpc...目的:探讨siRNA抑制TGFβ3诱发腭裂的机制。方法:使用不同剂量(200、400、800pmol)经脂质体LipofectamineTM2000包裹的TGFβ3 StealthTMRNAi于交配后(days post coitum,dpc)12天做孕鼠宫腔注射,等体积生理盐水注射作为空白对照。14dpc时取部分胎鼠腭突,RT-PCR及Western印迹检测腭突中TGFβ3、Smad2、BMP2的表达情况。16dpc时再取部分胎鼠腭突,观察腭突融合情况,免疫组织化学及免疫荧光观察胚鼠腭突的TGFβ3、Smad2、BMP2的表达情况。实验数据均以SPSS13.0软件包进行分析,多组间比较采用单因素方差分析,两组间比较采用t检验。结果:宫腔注射800pmol的StealthTMRNAi,可引起TGFβ3基因mRNA和蛋白表达水平明显降低,400pmol组能显著降低Smad2基因的mRNA和蛋白表达水平,但BMP2的表达情况未受明显影响。20%～60%的实验组小鼠胚胎腭突不能融合,形成腭隐裂,与空白对照组相比,其TGFβ3、Smad2的表达量较低,而BMP2的表达未见显著变化。结论:在12dpc给予小鼠宫腔注射TGFβ3 StealthTMRNAi,能有效沉默TGFβ3基因,20%～60%小鼠胚胎形成腭隐裂,同时Smad2基因表达下调,但对BMP2基因表达无明显影响。展开更多
Smads is a new gene family in transforming growth factor-β(TGF-β) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resu...Smads is a new gene family in transforming growth factor-β(TGF-β) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.展开更多
文摘目的:探讨siRNA抑制TGFβ3诱发腭裂的机制。方法:使用不同剂量(200、400、800pmol)经脂质体LipofectamineTM2000包裹的TGFβ3 StealthTMRNAi于交配后(days post coitum,dpc)12天做孕鼠宫腔注射,等体积生理盐水注射作为空白对照。14dpc时取部分胎鼠腭突,RT-PCR及Western印迹检测腭突中TGFβ3、Smad2、BMP2的表达情况。16dpc时再取部分胎鼠腭突,观察腭突融合情况,免疫组织化学及免疫荧光观察胚鼠腭突的TGFβ3、Smad2、BMP2的表达情况。实验数据均以SPSS13.0软件包进行分析,多组间比较采用单因素方差分析,两组间比较采用t检验。结果:宫腔注射800pmol的StealthTMRNAi,可引起TGFβ3基因mRNA和蛋白表达水平明显降低,400pmol组能显著降低Smad2基因的mRNA和蛋白表达水平,但BMP2的表达情况未受明显影响。20%～60%的实验组小鼠胚胎腭突不能融合,形成腭隐裂,与空白对照组相比,其TGFβ3、Smad2的表达量较低,而BMP2的表达未见显著变化。结论:在12dpc给予小鼠宫腔注射TGFβ3 StealthTMRNAi,能有效沉默TGFβ3基因,20%～60%小鼠胚胎形成腭隐裂,同时Smad2基因表达下调,但对BMP2基因表达无明显影响。
基金This work was supported by Hi-Tech Research and Development Program of China (102-08-08-02 ) National Science Fund for Distinguished Young Scholars ( 30025028 ).
文摘Smads is a new gene family in transforming growth factor-β(TGF-β) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.
