Milanetto, Martina (2008) Caratterizzazione dell'espressione e della funzione delle Emiline in topo ed in zebrafish. [Tesi di dottorato]
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Emilins are a family of glycoproteins of the extracellular matrix with common structural organization and containing a characteristic N-terminal cysteine-rich domain. During my PhD, I studied expression and function in zebrafish and in mice of the corresponding genes, and I focused my attention on Emilin3, coding for the only component of the family lacking the C-terminal gC1q domain.
As concerns my work on zebrafish, by database searching we identified and cloned eight members of this gene family. Their expression pattern during embryonic development, analyzed by whole-mount in situ hybridization, is characteristic and partially overlapping with expression of Emilins in mice. The most interesting pattern is the one of Emilin-3: while other Emilin genes reveal a mesenchymal or cardiovascular expression profile, the two zebrafish genes coding for Emilin-3 are abundantly expressed at early stages in the notochord and in the floor plate and at later stages in the branchial arches. This suggests that Emilin-3 may play a distinct role in the extracellular matrix, in comparison with other members of Emilin family. To gain insight into the function of these genes in vivo, we started functional studies by in situ hybridization of embryos in which relevant molecular pathways are blocked by treatment with specific drugs and by microinjection of mRNA and morfolino oligonucleotides in fertilized oocytes.
Concerning the studies in mice, these experiments represented a large part of my PhD work and they were concentrated on the gene coding for Emilin-3. In the past years, targeted Emilin3 gene inactivation in murine embryonic stem (ES) cells was already undertaken in the laboratory where I performed my PhD work. However, generation of Emilin3 knockout mice was unsuccesful and these previous attempts revealed that, unlike other Emilins, targeted inactivation of the Emilin3 gene is particularly difficult. The frequency of homologous recombination of this gene is extremely low and transmission of the inactivated allele to the F1 generation was not reached, probably also due to karyotypic instability of targeted ES cell clones. Therefore, I carried out a new Emilin3 gene targeting approach, performed by means of a large scale experiment with a new ES cell line. I transfected euploid R1 ES cells with a Emilin3 targeting construct and, after double positive-negative selection, 1505 clones were isolated; 995 of these individual cell clones were further expandend and investigated for identifying clones in which a correct homologous recombination event had occurred. With such large number of clones, molecular analysis by Southern blotting, a standard but time- and money-consuming procedure, was not an easily affordable screening method for detecting rare homologus recombinant clones. Therefore, I set up an optimal screening procedure by PCR. Six positive ES clones were identified, and after additional characterization four of them were used for generation of chimeric mice by microinjection of ES cells into host blastocysts and implant in foster females. By crossing these chimeric mice, I obtained for two independent clones heterozygous and finally homozygous Emilin3 knockout mice, and I started to undertake the phenotypic analysis of Emilin-3 null mice. Finally, I also performed a detailed karyotypic analysis of targeted Emilin3 ES clones introduced in vivo, in comparison to the original ES cell line and other mutant ES cell clones, with the aim of correlating the karyotype of ES cells with their ability to transmit the inactivated allele to the germ-line.
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