Molecular cloning of transcription factor for odontblast differentiation

利用退化引物RT-PCR从切牙髓RNA中克隆出锌指转录因子G23/Gli5新成员。小鼠G23编码的5个锌指结构域与Gli1、Gli2和Gli3的氨基酸序列同源性接近65%。Northern blot分析显示G23/Gli5在牙髓、肾脏和睾丸中表达。原位杂交显示，G23/Gli5在交配后10.0天（dpc）首次在前肢和后肢芽中广泛检测到。10.5 dpc时，在鳃弓、肢芽、颅面缘和尾腹部表达。随后在须囊、椎间盘、肾脏和睾丸中表达。在牙胚中，G23/Gli5在钟状牙乳头中表达，提示其可能在成牙细胞分化过程中起抑制作用。我们对G23/Gli5进行了靶向破坏。第二个外显子，位于5'-未翻译区至第3锌指DNA结合域850bp处，包含起始密码子，ATG被LacZ polyA和lox-neo-polyA盒式取代。同源重组经Southern blot和PCR证实。纯合子是完全活的，似乎没有任何明显的表型。G23/Gli5和Gli1家族成员的互补表达模式表明G23/Gli5和Gli1家族成员具有冗余功能。我们现在正在分析双纯合子，以发现G23/Gli5在牙齿发育过程中的功能。

We have cloned a new member of the zinc finger transcription factor, G23/Gli5 from incisor pulp RNA by RT-PCR using degenerate primers. The five zinc finger domain encoded by mouse G23 has almost 65% amino acid sequence homology with Gli1, Gli2 and Gli3. Northern blot analysis revealed G23/Gli5 is expressed in dental pulp, kidney and testis. In situ hybridization of sections and while mount embryos demonstrated G23/Gli5 is first detected diffusely in forelimb and hindlimb bud at 10.0 days post coitus (dpc). At 10.5 dpc, it is expressed in the branchial arches, limb bud, craniofacial border, and ventral part of the tail. Later, it is expressed in whisker follicle, intervertebral disc, kidney, and testis. In tooth germ, G23/Gli5 is expressed in dental papillae at bell stage, suggesting that it might function as a repressor during odontoblast differentiation.We made targeted disruption of G23/Gli5. The second exon, 850bp from 5'-untranslated region to 3rd zinc finger DNA binding domain, containing the start codon, ATG was replaced by LacZ polyA and the lox-neo-polyA cassette. Homologous recombination was confirmed by Southern blot and by PCR. The homozygotes were fully viable and did not seem to have any conspicuous phenotype. The complementary expression patterns of G23/Gli5 and Gli1 family members suggest the redundant function of G23/Gli5 and Gli1 members. We are now analyzing the double homozygotes to find the function of G23/Gli5 during tooth development.