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Generation of model mice for brain tumors using conditional knockout technique

使用条件敲除技术产生脑肿瘤模型小鼠

基本信息

批准号：
17390395
负责人：
USUI Hiroshi
金额：
$ 10.52万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

The purpose of this research is generating model mice for brain tumors, using conditional knockout technique to destroy the functions of tumor-suppressor genes selectively in brain cells. We produced five lines of genetically modified mice, by use of the homologous recombination vector DNAs and mouse ES cell line RENKA. The five lines included 2 kinds of "flox" mice in which frame-shift exon of tumor-suppressor gene Ptc or Nf1 was flanked with recombination target sequence loxP, and 2 kinds of "knock-in" mice in which Cre recombinase was introduced in the translation initiation site of Nestin or Tubb3 gene, marker genes for neural cells. We also produced Nf1-flox; P53-null mice in which a tumor suppressor gene P53 located nearly in Nf1 gene was destroyed. Crossbreeding of the "flox" mice and "knock-in" mice successfully generated the mice in which tumor suppressor gene was destroyed selectively in brain cells by the Cre-loxP recombination system.Among the mice obtained, brain tumors which arose from the cells in cerebellar cortex were observed with frequency of 50% (2/4)in Ptc-flox;Tubb3-Cre hetero mice before 5 months of age. The tumor cells consisted of undifferentiated cells which hardly expressed mature marker proteins and resembled those of human medulloblastomas in histology. On the other hand, no brain tumor was observed in Nf1-flox;Neatin-Cre hetero nor Nf1-flox;P53-null;Neatin-Cre hetero mice within 6 months of age, suggesting the necessity of generating homo knockout mice for the Nf1 and P53 gene.These results indicate that mutation of Ptc gene is one of the causative events of medulloblastoma, and, therefore, we were able to contribute to clarification of etiology of the medulloblastoma. In addition, the Ptc-flox;Tubb3-Cre hetero mice may work as model mice of medulloblastoma which is useful to develop new therapeutic drugs or protocols.

本研究的目的是建立脑肿瘤模型小鼠，利用条件敲除技术选择性地破坏脑细胞中肿瘤抑制基因的功能。利用同源重组载体dna和小鼠ES细胞系RENKA制备了5株转基因小鼠。这5个系包括2种肿瘤抑制基因Ptc或Nf1的移框外显子侧接重组靶序列loxP的“flox”小鼠，以及2种在神经细胞标记基因Nestin或Tubb3基因的翻译起始位点引入Cre重组酶的“knock-in”小鼠。我们还生产Nf1-flox；肿瘤抑制基因P53位于Nf1基因附近的P53缺失小鼠被破坏。将“flox”小鼠与“knock-in”小鼠杂交，成功地产生了Cre-loxP重组系统选择性破坏脑细胞中肿瘤抑制基因的小鼠。在所获得的小鼠中，Ptc-flox以50%（2/4）的频率观察到小脑皮质细胞产生的脑瘤；5个月前的Tubb3-Cre异种小鼠。肿瘤细胞由未分化的细胞组成，几乎不表达成熟的标记蛋白，在组织学上与人髓母细胞瘤相似。另一方面，Nf1-flox未见脑肿瘤；Neatin-Cre异质或Nf1-flox；P53-null；6月龄以内的Neatin-Cre异型小鼠，提示产生Nf1和P53基因敲除小鼠的必要性。这些结果表明Ptc基因突变是成神经管细胞瘤的致病事件之一，因此我们能够有助于澄清成神经管细胞瘤的病因。此外，Ptc-flox；Tubb3-Cre异种小鼠可以作为成神经管细胞瘤的模型小鼠，这对开发新的治疗药物或治疗方案具有重要意义。