Assessment of individual functions of Myc family genes

为了检查N-myc和L-myc基因之间功能冗余的可能性，我们产生了N^<+/->L^<-/->以及N-myc和L-myc双突变小鼠（N^<-/->L^<-/->）。N-myc和L-myc双突变体胚胎显示出与仅在小鼠中切除N-myc（N^<-/->L^<+/+>或N^<-/->L^<+/>）相似的总体表型。为了鉴定N-myc调控靶基因，我们在10.5dpc时从突变体胚胎的总cDNA中减去野生型cDNA，反之亦然，并分离出突变体中下调的mRNA或相反组合中上调的mRNA的cDNA。对N-myc下游基因之一的Ndr 1进行了详细的研究。Ndr 1基因启动子直接被N-myc和Max异源二聚体复合物抑制。N-myc和c-myc在胚胎发育过程中的作用是相互制约的，因此我们将c-myc基因的编码区引入N-myc基因座，使其受N-myc基因的调控。在32只胚胎干细胞“父代”幼仔中未观察到杂合子后代。这是一个强有力的迹象表明，表达来自N-myc基因座的c-myc的胚胎在子宫内死亡或甚至可能不会产生。

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.