Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

Autor: Michael R. A. Mowat, Cordula Buse, Mohammad Golam Sabbir, Nichola Wigle, Yuan Gu, Geoffrey G. Hicks, Shauna Loewen
Rok vydání: 2010
Předmět:
Male
rho GTP-Binding Proteins
Gene isoform
Genetically modified mouse
RHOA
Genotype
Physiology
Blotting
Western
Mutant
Plant Science
General Biochemistry
Genetics and Molecular Biology
Cell Line
Mice
Exon
Structural Biology
SNAP23
Animals
Protein Isoforms
Promoter Regions
Genetic
lcsh:QH301-705.5
Gene
Cells
Cultured
Cytoskeleton
Ecology
Evolution
Behavior and Systematics
Agricultural and Biological Sciences(all)
biology
Biochemistry
Genetics and Molecular Biology(all)
Reverse Transcriptase Polymerase Chain Reaction
Tumor Suppressor Proteins
GTPase-Activating Proteins
Days post coitum
Cell Biology
DNA Methylation
Blotting
Northern
Embryo
Mammalian
Immunohistochemistry
Molecular biology
Mice
Inbred C57BL
lcsh:Biology (General)
biology.protein
Female
rhoA GTP-Binding Protein
General Agricultural and Biological Sciences
Nucleic Acid Amplification Techniques
Research Article
Developmental Biology
Biotechnology
Zdroj: BMC Biology
BMC Biology, Vol 8, Iss 1, p 17 (2010)
ISSN: 1741-7007
DOI: 10.1186/1741-7007-8-17
Popis: Background 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. Results 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. Conclusions 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.
Databáze: OpenAIRE
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