Assessment of individual functions of Myc family genes

为了验证N-myc和L-myc基因之间功能冗余的可能性，我们产生了N^&lt；+/-&gt；L^&lt；-/-&gt；以及N^&lt；-/-&gt；N^&lt；-/-&gt；L^&&gt；N-myc和L-myc双突变胚胎的总体表型与仅在小鼠中去除N-myc相似(N^&lt；-/-gt；L^&lt；+/+&gt；或N^&lt；-/-&gt；L^&lt；+/&gt；)。结果表明，L-myc对胚胎发育不是必需的，并否定了N-myc和L-myc基因之间功能冗余的观点。为了鉴定N-myc调控靶基因，我们从10.5dpc的野生型cdna中减去了突变胚胎的总cDNA，反之亦然，并分离了突变体中下调表达的基因或相反组合中上调表达的mRNAs。对N-myc下游基因ndrl进行了详细的研究。N-myc和Max异源二聚体复合体直接抑制了ndrl基因的启动子。在不同的胚胎器官中，N-myc和ndrl的表达呈负相关，表明N-myc对ndrl的抑制是胚胎发育过程中的一种调节。为了解决N-myc和c-myc是否有重叠的功能，我们在N-myc基因座上引入了c-myc编码区，使其受N-myc的调控。在32只ES细胞“父亲”仔鼠中没有观察到杂合子后代。这是一个强烈的迹象，表明从N-myc基因座表达c-myc的胚胎在子宫中死亡，甚至可能不会产生。

To check the possibility of functional redundancy between the N-myc and L-myc genes, we generated N^<+/->L^<-/-> as well as N-myc and L-myc double mutant mice (N^<-/->L^<-/->). N-myc and L-myc double mutant embryos showed an overall phenotype similar to ablating N-myc only in mice (N^<-/->L^<+/+> or N^<-/->L^<+/>). The results indicate that L-myc is not essential for embryonic development, and negates the notion of redundant functions between the N-myc and L-myc genes.To identify N-myc regulatory target genes we subtracted total cDNAs of mutant embryo from wild type cDNAs at 10.5 dpc and vice versa, and isolated cDNA of down-regulated mRNA in the mutants or of up-regulated mRNA species in the opposite combination. One of the N-myc downstream genes, Ndrl was studied in detail. The promoter of Ndrl gene was directly repressed by N-myc and Max heterodimer complex. In various embryonic organ rudiments an inverse relationship was found between the expression of N-myc and Ndrl, indicating that repression of the latter by the former is a regulation operating in embryogenesis.To address whether N-myc and c-myc have overlapping functions, we have introduced the c-myc coding region into the N-myc locus so that it comes under the regulation of N-myc. No heterozygous offspring were observed among 32 ES cell "fathered" pups. This is a strong indication that the embryos expressing c-myc from the N-myc locus die in utero or may not even be generated.