Abnormal gene expressions in the Gli3 and Shh of the mouse homolog of GCPS, Pdn

遗传性多趾/大脑畸形小鼠（Pdn/Pdn）的表型与Greig头多趾综合征（GCPS）相似，其负责基因为GLI3。在Pdn/Pdn中观察到Gli3基因表达的抑制。因此，Pdn/Pdn的负责基因一直被认为是Gli3，但突变点尚未明确。本课题确定Pdn小鼠Gli3基因内含子3的41286/41287位置上插入了一个转座子的5442。该转座子两端有317bp长的末端重复序列。在插入点附近的内含子3上构建正向引物和反向引物，在转座子上构建正向引物。然后用这些引物进行PCR产物鉴别+/+、Pdn/+、Pdn/Pdn胚胎。利用这种快速PCR方法，我们可以确定幼胚的基因型。在胚胎期和新生儿期，Gli3基因在Pdn/+中的表达被抑制到60%左右，在Pdn/Pdn中的表达被抑制到20-30%，但没有观察到基因表达的阶段依赖性。Shh被认为是Gli3的拮抗剂。因此，我们推测Shh可能在Gli3基因表达被抑制的Pdn/Pdn小鼠胚胎中过表达。基因型间Shh表达差异仅在妊娠第9天存在。

Phenotype of genetic polydactly/archinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), whose responsible gene is GLI3. Suppression of Gli3 gene expression had been observed in Pdn/Pdn. So, the responsible gene of Pdn/Pdn has been considered to be Gli3, but mutation point has not been demarcated.In this research project, it was determined that 5442 of a transposon was inserted in the position 41286/41287 of intron 3 of Gli3 gene in Pdn mouse. This transposon has 317-bp long terminal repeats in both ends. Forward and reverse PCR primers were constructed in the intron 3 near the insertion point, and forward primer in the transposon was also a constructed. Then we could discriminate +/+, Pdn/+, Pdn/Pdn embryos with the PCR product by these primers. Using this rapid PCR method, we can determine the genotypes of the young embryos.Gli3 gene expression in Pdn/+ was suppressed to about 60% for +/+, and that in Pdn/Pdn was 20-30% for +/+ through all the embryonic and neonatal periods examined, but stage dependency of gene expression was not observed. Shh has been considered to be antagonist of Gli3. So, we speculated that Shh would be overexpressed in the Pdn/Pdn mouse embryos whose Gli3 gene expression was suppressed. Shh expression was different among genotypes only by on day of 9 of gestation.