Generation of model mice for brain tumors using conditional knockout technique

本研究的目的是利用条件性基因敲除技术选择性地破坏脑细胞中肿瘤抑制基因的功能，从而建立脑肿瘤模型小鼠。我们利用同源重组载体DNA和小鼠ES细胞系RENKA获得了5个转基因小鼠系。这5个品系包括2种以重组靶序列loxP为侧翼的抑癌基因PTC或Nf1的移码外显子，以及2种在神经细胞的标志基因Nestin或TUBB3的翻译起始点引入Cre重组酶的“敲入”小鼠。我们还获得了NF1-FLOX；P53缺失的小鼠，其中位于NF1基因附近的抑癌基因P53被破坏。利用Cre-loxP基因重组系统成功地将“FLOX”小鼠与“敲入”小鼠杂交，获得了Cre-loxP重组系统选择性破坏脑细胞中肿瘤抑制基因的小鼠，其中PTC-FLOX、TUB3-Cre异种小鼠在5月龄前发生脑瘤的比例为50%(2/4)。肿瘤细胞由未分化的细胞组成，几乎不表达成熟标志蛋白，在组织学上与人髓母细胞瘤相似。另一方面，在Nf1-Flox、Neatin-Cre异源或Nf1-Flox、P53-Null、Neatin-Cre异种小鼠中，在6个月龄内未观察到脑肿瘤，提示有必要产生NF1和P53基因的同源敲除小鼠。这些结果表明，PTC基因突变是髓母细胞瘤的致病事件之一，因此，我们能够为阐明髓母细胞瘤的病因做出贡献。另外，PTC-FLOX；TUBB3-Cre异种小鼠可作为髓母细胞瘤的模型小鼠，为开发新的治疗药物或方案提供依据。

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.