Gasca S, Hill D P, Klingensmith J, Rossant J
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Dev Genet. 1995;17(2):141-54. doi: 10.1002/dvg.1020170206.
We have used a gene trap (GT) vector and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. GT vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. In this paper we describe a GT insertion which displays a very restricted pattern of expression in the gastrulating embryo. beta-Galactosidase activity was first detected at 7.5 days post-coitum (E7.5) in the node region of the embryo and extended to the midline structures at E8.0. At E9.5 expression was restricted to the floor plate, the notochord, the roof of the gut, and the liver anlage. Expression appeared in the somites at E10.0 and later became more widespread. We used rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to clone a partial 360 base pair (bp) cDNA representing an endogenous sequence and containing an open reading frame (ORF) fused in frame to the lacZ reporter gene. The sequence showed no homology to any known protein or protein domain. An overlapping 1,200 bp fragment from a wild-type cDNA library was cloned and it detected the same pattern of expression as the reporter gene in E7.5, E8.5, and E9.5 wild-type embryos. It hybridized to a 5.4 kb lacZ fusion transcript and to an endogenous transcript of 6.5 kb. The gene was mapped to chromosome 11 and was named cordon-bleu (cobl). No phenotype was detected in mice homozygous for the insertion. However, the insertion may not cause a complete disruption of the gene function. The pattern of expression of cobl is very similar to that of hepatic nuclear factor 3 beta (HNF3 beta) and sonic hedgehog (Shh), both of which are involved in axial patterning. Therefore, the product of the cobl gene may also prove to be an important component of the genetic pathway regulating vertebrate axis formation.
我们利用基因捕获（GT）载体和胚胎干细胞（ES）细胞嵌合体，筛选β-半乳糖苷酶报告基因插入到在小鼠早期胚胎发育过程中受到时空调控的转录单位的情况。GT载体既能作为报告基因又能作为诱变剂，此前已被用于分离对小鼠发育至关重要的新基因。在本文中，我们描述了一个GT插入事件，其在原肠胚形成期胚胎中呈现出非常局限的表达模式。β-半乳糖苷酶活性在胚胎期7.5天（E7.5）首次在胚胎的节点区域被检测到，并在E8.0时扩展到中线结构。在E9.5时，表达局限于底板、脊索、肠顶和肝脏原基。在E10.0时，表达出现在体节中，随后变得更加广泛。我们使用cDNA末端快速扩增-聚合酶链反应（RACE-PCR）克隆了一个360个碱基对（bp）的部分cDNA，它代表一个内源序列，并且包含一个与β-半乳糖苷酶报告基因读框融合的开放阅读框（ORF）。该序列与任何已知蛋白质或蛋白质结构域均无同源性。从野生型cDNA文库中克隆了一个1200 bp的重叠片段，它在E7.5、E8.5和E9.5野生型胚胎中检测到与报告基因相同的表达模式。它与一个5.4 kb的β-半乳糖苷酶融合转录本以及一个6.5 kb的内源转录本杂交。该基因被定位到11号染色体，并被命名为蓝带（cobl）。在插入纯合的小鼠中未检测到表型。然而，该插入可能并未导致基因功能的完全破坏。cobl的表达模式与肝细胞核因子3β（HNF3β）和音猬因子（Shh）的表达模式非常相似，这两者都参与轴向模式形成。因此，cobl基因的产物可能也被证明是调节脊椎动物轴形成的遗传途径的一个重要组成部分。
