Construction of model mouse of the Beckwith-wiedemann syndrome

CDK抑制剂p57^<KIP2>是导致Beckwitn-Wiedemann综合征（BWS）的基因之一，其位点位于染色体11p15.5。为了证实该突变体在细胞和动物水平上的表型，1)我们将该突变体转染到细胞中，观察其生化和形态学效应；2)我们也建立了转基因小鼠作为BWS模型。将携带突变体p57^<KIP2> cDNA的腺病毒载体转染G401和RD细胞。这些细胞不表达内源性p57KIP2。突变体为Ad/KIP2-1-8 （p57^<KIP2>的C-ter突变）。控制是Ad/KIP2-1（野生p57^<KIP2>）和Ad/LacZ。转染野生p57^<KIP2> cDNA的RD细胞表达该蛋白。Western blotting和p57KIP2抗体染色证实了这一点。然而，野生p57^<KIP2 bb0未观察到对细胞生长的特异性抑制。抑制水平与突变体p57^<KIP2>相似。接下来，我们尝试构建携带突变体p57^<KIP2>的转基因小鼠作为模拟BWS的动物模型。利用pRc/CVM质粒载体获得了重组人cDNA的高表达。这些包括pDR2-1（野生），pDR2-1-6 （CDK抑制结构域突变）和pDR2-1-8 （QT结构域突变）。结果显示，所有出生的小鼠都是非转基因的，这表明该基因的过度表达可能是有毒的。为了克服这种情况，将来有必要采用诸如Cre/Lox系统之类的条件转基因。

The CDK inhibitor, p57^<KIP2> is one of the gene responsible for Beckwitn-Wiedemann syndrome (BWS) of which locus is located on chromosome 11p15.5. To confirm the phenotype of this mutant at the cellular level and the animal, 1) we transfected the mutant to the cell and observed the biochemical and morphorogical effect, 2) and also created the transgenic mouse as a model of BWS.Adenovirus vector carrying mutant p57^<KIP2> cDNA was transfected to the G401 and RD cells. These cells do not express the endogenous p57KIP2. The mutant is Ad/KIP2-1-8 (mutaion at the C-ter of p57^<KIP2>). Controls are Ad/KIP2-1 (wild p57^<KIP2>) and Ad/LacZ.RD cell transfected with wild p57^<KIP2> cDNA expressed the protein. This was confirmed by Western blotting and by the staining with p57KIP2 antibody. However, the specific repression of the cell growth was not observed by the wild p57^<KIP2>. The level of the repression was similar with that of the mutant p57^<KIP2>. Next, we tried to create the transgenic mouse carrying mutant p57^<KIP2> as animal model to mimic BWS.pRc/CVM plasmid vector was used to obtain the high expression of human cDNA recombinant. These include pDR2-1 (wild), pDR2-1-6 (mutation of the CDK inhibitory domain) and pDR2-1-8 (mutaion at the QT domain). The obtained result showed all mice born were non-transgenic, suggesting the overexpression of this gene might be toxic. Conditional transgenics such as Cre/Lox system will be necessary to overcome this situation in the future.