Molecular cloning of transcription factor for odontblast differentiation

我们用简并引物通过RT-PCR从切牙牙髓RNA中克隆了一个锌指转录因子G23/Gli 5。小鼠G23编码的5个锌指结构域与Gli 1、Gli 2和Gli 3的氨基酸序列同源性接近65%。北方印迹分析显示G23/Gli 5在牙髓、肾脏和睾丸中表达。原位杂交结果表明，G23/Gli 5在交配后10.0天（dpc）的前肢和后肢芽中首先被广泛检测到。在10.5 dpc时，它在鳃弓、肢芽、颅面缘和尾腹侧部分表达。后来，它在须囊、椎间盘、肾脏和睾丸中表达。在牙胚中，G23/Gli 5在钟状期的牙乳头中表达，提示其可能在成牙本质细胞分化过程中起抑制作用。第二个外显子，从5 '-非翻译区到第三个锌指DNA结合结构域的850 bp，含有起始密码子ATG，被LacZ polyA和lox-neo-polyA盒取代。通过Southern印迹和PCR证实同源重组。纯合子是完全可行的，似乎没有任何明显的表型。G23/Gli 5和Gli 1家族成员的互补表达模式表明G23/Gli 5和Gli 1成员的冗余功能。我们现在正在分析双纯合子，以发现G23/Gli 5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.