Li Ling, Zhu Ji, Tu Qisheng, Yamauchi Masato, Sodek Jaro, Karsenty Gerard, Tang Jean, Chen Jake
Division of Oral Biology, Tufts School of Dental Medicine, Boston, Massachusetts 02111, USA.
J Bone Miner Res. 2005 Aug;20(8):1403-13. doi: 10.1359/JBMR.050316. Epub 2005 Mar 21.
To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene.
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.
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.
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 approximately 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.
为了研究体内骨骼发育，建立了一种转基因小鼠模型，其中由骨唾液蛋白（BSP）启动子驱动的禽逆转录病毒受体（TVA）基因在骨骼组织中选择性表达。通过显示感染表达突变Cbfa1/Runx2基因的病毒后BSP表达受到抑制以及骨骼和牙齿形成延迟，验证了该模型。
禽逆转录病毒（TVA）受体的组织特异性表达可用于在体内有效靶向基因的异位表达。为了确定这种方法在特定发育阶段成骨分化和骨骼形成研究中的用途，构建了在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，分析显性负性形式的Cbfa1/Runx2转录因子对骨骼形成的影响。
我们首先生成了转基因小鼠（BSP/TVA），其中TVA基因在4.9 kb小鼠BSP启动子的控制下表达。通过逆转录聚合酶链反应（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，并与内源性BSP基因的表达进行比较。将编码DNA结合域的396 bp突变Cbfa1/Runx2（Cbfa1mu）片段克隆到RCASBP（A）病毒载体中，用于感染新生BSP/TVA小鼠。
转基因小鼠中TVA受体mRNA和蛋白的表达与内源性BSP的表达一致。用Cbfa1mu-RCASBP（A）载体全身感染4天后，RT-PCR分析显示胫骨和下颌骨中BSP mRNA的表达几乎被消除，而颅骨中则降低了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BSP的表达降低了45%，而颅骨膜性骨中BSP的表达降低了约15%。然而，4周和8周后，任何骨组织中BSP的表达几乎没有变化。相比之下，仅在病毒转染9天后在三块骨中观察到骨桥蛋白表达降低。组织形态学检查显示，一些感染突变Cbfa1的小鼠骨骼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部位，为研究体内与骨骼形成相关的分子事件提供了一个独特的系统。
