Manitoba Institute of Cell Biology, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, MB R3E 0V9, Canada.
BMC Biol. 2010 Mar 3;8:17. doi: 10.1186/1741-7007-8-17.
The Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.
In addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay.
The mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.
Dlc1（肝癌缺失 1 号）肿瘤抑制基因编码一种 RhoGTPase 激活蛋白，存在于多种肿瘤类型中失活。已经描述了几种转录异构体，但每种形式的功能意义和组织分布目前了解甚少。此外，不同哺乳动物物种之间报告的异构体和剪接变体数量存在差异。为了更好地了解小鼠中 Dlc1 基因的不同变体的数量和功能，我们进行了详细的分析。进行了广泛的 3' RACE 实验，以鉴定小鼠中所有可能的 Dlc1 异构体和剪接变体。此外，我们生成了一种基因捕获小鼠，该小鼠靶向其中一种异构体，以研究其生物学功能。还研究了该基因捕获插入对其他异构体剪接的影响。
除了已知的 Dlc1 的 6.1 和 6.2 Kb 转录本外，我们的研究还揭示了小鼠中存在一种新的 7.6 Kb 转录本，该转录本与人类的 7.4 Kb（KIAA1723）cDNA 转录本相对应。一种基因捕获胚胎细胞系，其插入 6.1 Kb 转录本的外显子 1 和 2 之间，被用于生成转基因小鼠。该系显示出捕获的异构体表达显著降低。然而，没有观察到其他异构体的表达减少。携带基因捕获等位基因的杂合小鼠表型正常，但纯合突变胚胎在妊娠后 10.5 天以上不能存活。Dlc1gt/gt 胚胎在大脑、心脏和胎盘血管中出现缺陷。从 Dlc1 缺失胚胎的无血清培养的小鼠胚胎细胞中，RhoA 活性升高，肌动蛋白丝和焦点粘连的组织发生改变。在体外划痕试验中，Dlc1 缺陷细胞的伤口闭合也增加。
小鼠有三种主要的 Dlc1 基因转录异构体，在各种组织中表达不同。6.1 Kb 转录本外显子 1 的小鼠 gt 导致 Dlc1 蛋白表达呈低等位基因表型，纯合条件下表现出胚胎致死表型，表明该异构体在小鼠发育中起主要作用。Dlc1 缺陷细胞表现出改变的细胞骨架结构、增加的 RhoA 活性和细胞迁移。
