Construction of model mouse of the Beckwith-wiedemann syndrome

CDK抑制基因p57^&lt；KIP2&gt；是Beckwitn-Wiedemann综合征(BWS)的致病基因之一，其基因座位于染色体11p15.5。为了在细胞和动物水平上确定突变体的表型，1)将突变体导入细胞，观察其对细胞生化和形态的影响；2)将携带突变体p57^&lt；KIP2&gt；cDNA腺病毒载体导入G401和RD细胞，建立BWS模型。这些细胞不表达内源性p57Kip2。该突变体为Ad/KIP2-1-8(p57^&lt；Kip2&gt；C位突变)。对照组为野生型p57；KIP2-1(野生型p57；KIP2)和野生型p57；KIP2&gt；转基因的Ad/LacZ.RD细胞。Western blotting和p57Kip2抗体染色证实了这一点。然而，野生型p57^&lt；KIP2&gt；没有观察到对细胞生长的特异性抑制。抑制水平与突变体p57^&lt；KIP2&gt；相似。接下来，我们尝试建立携带突变体p57^&lt；KIP2&gt；的转基因小鼠，作为模拟BWS的动物模型。其中包括pDR2-1(野生型)、pDR2-1-6(CDK抑制区突变)和pDR2-1-8(QT区突变)。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。有条件的转基因，如Cre/Lox系统，将是未来克服这种情况的必要手段。

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.