An in vivo model to study osteogenic gene regulation: Targeting an avian retroviral receptor (TVA) to bone with the bone sialoprotein (BSP) promoter
An in vivo model to study osteogenic gene regulation: Targeting an avian retroviral receptor (TVA) to bone with the bone sialoprotein (BSP) promoter
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DOI：
10.1359/jbmr.050316
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发表时间：
2005-08-01
影响因子：
6.2
通讯作者：
Chen, J
中科院分区：
文献类型：
--
作者：
Li, L;Zhu, J;Chen, J
简介：禽逆转录病毒（TVA）受体的组织特异性表达可用于有效地靶向体内基因的异位表达。为了确定该方法在特定发育阶段的成骨分化和骨形成研究中的用途，产生了在 5-kb 骨唾液蛋白 (BSP) 启动子控制下表达 TVA 受体的转基因小鼠。首先分析小鼠 TVA 基因的组织特异性表达，然后在感染病毒构建体后，分析显性失活形式的 Cbfa1/Runx2 转录因子对骨形成的影响。 材料和方法：我们首先生成转基因小鼠 (BSP/TVA)，其中 TVA 基因在 4.9-kb 小鼠 BSP 启动子的控制下表达。通过RT-PCR、原位杂交和免疫组织化学分析TVA基因的组织特异性表达，并与内源BSP基因的表达进行比较。将编码DNA结合域的突变Cbfa1/Runx2（Cbfa1mu）的396 bp片段克隆到RCASBP（A）病毒载体中，用于感染新生BSP/TVA小鼠。结果与结论：转基因小鼠中TVA受体mRNA和蛋白的表达与内源BSP的表达一致。 Cbfa1mu-RCASBP (A) 载体全身感染四天后，RT-PCR 分析显示，胫骨和下颌骨中 BSP mRNA 的表达几乎消失，而颅骨中 BSP mRNA 的表达减少了 30%。 9天后，与感染空RCASBP载体的对照动物相比，胫骨和下颌骨中的BSP表达减少了45%，而颅盖膜骨中的BSP表达减少了约15%。然而，4周和8周后，任何骨组织中的BSP表达几乎没有变化。相比之下，病毒转染后 9 天，三块骨头中才观察到骨桥蛋白表达减少。组织形态学检查显示，一些感染 Cbfa1 突变的小鼠的骨形成和牙齿发育延迟。这些研究表明，BSP/TVA 转基因小鼠可用于将基因靶向成骨位点，为研究与体内骨形成相关的分子事件提供了独特的系统。
Introduction: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation.Materials and Methods: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice.Results and Conclusion: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased similar to 15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.