Objective: The aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered.
Methods: The C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development.
Results: TTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry.
Conclusion: The recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.
目的: 建立腭裂相关基因甲状腺转录因子-2（TTF-2）转基因小鼠模型，利用转基因动物模型研究TTF-2基因的活动规律和表达模式发生变化时对腭突发育过程产生的影响。
方法: 采用聚合酶链反应法扩增C57BL/6J小鼠基因组TTF-2基因，将其定向插入pBROAD3-mcs载体，构建重组表达质粒pBROAD3-TTF-2，利用显微注射技术把线性化表达载体注射到受精卵的雄原核中，建立TTF-2转基因小鼠。利用特异引物聚合酶链反应和Southern blot方法鉴定转基因小鼠的基因型，采用免疫组织化学法检测TTF-2基因在转基因小鼠腭突组织中的表达。
结果: 共注射原核期受精卵982枚，发育为2-细胞胚胎的580枚，均移植入48个假孕受体昆明白小鼠中，得到胎鼠68只。提取基因组DNA，发现有13只转基因阳性的胎鼠。免疫组织化学法检测显示TTF-2在转基因小鼠腭突中持续表达。
结论: 通过显微注射法使外源基因pBROAD3-TTF-2整合入小鼠基因组中，成功建立了腭突持续表达TTF-2的转基因小鼠模型，其表现型为腭裂。