engleski

Targeted gene inactivation of murine Emilin-3, a novel component of extracellular matrix

The EMILINs (Elastin Microfibril Interface Located proteINs) are glycoproteins of the extracellular matrix (ECM), associated with elastic fibers and characterized by the EMI, cysteine-rich N-terminal domain. Emilin homotrimers are formed by the stable interactions of their C-terminal gC1q domain. Emilin-3 (formerly named Emilin-T) lacks the gC1q domain and thus is probably incapable of forming multimers. It is possible that Emilin-3 plays a different role in the ECM compared to other members of Emilin family, which are considered to play a key role in the process of elastogenesis and in mediating cell-ECM interactions. To gain insight into the function of Emilin-3, the Emilin3 gene was inactivated by gene targeting technology. By this approach, a gene targeting construct, containing a portion of the Emilin3 gene and positive/negative selectable markers, was introduced into ES cells by electroporation. During our first attempt to inactivate Emilin3 gene, correctly targeted ES clones were not found (382 ES clones analyzed in total). It was discovered that neo cassette (a part of targeting construct) contained a point mutation that substantially reduced neomycin phosphotransferase activity. Therefore, a new targeting vector was prepared by using another neo cassette (originated from pHM2) and then it was introduced into ES cells. Out of 739 analyzed ES clones, 12 were found to contain a correctly inactivated Emilin3 gene. After additional Southern and northern blot analysis of these selected ES clones, chimeric (G0) mice were produced by microinjection of 3 correctly targeted clones (E3B-589, E3B-849 and E3B-856) into host C57BL/6J blastocysts and subsequently implanted into CD1 foster mothers. Although chimeric mice were obtained for all three ES clones, the contribution of ESderived cells to host tissues was relatively low, being of 50% or lower. Furthermore, selected chimeras that were crossed with C57BL/6J mice in order to obtain F1 generation, did not transmit the inactivated ES-derived allele in their germ-line, and thus no heterozygous (Emilin3+/–) offspring were obtained. In fact, all animals obtained in the F1 generation were completely black coat colored, thus indicating their C57BL/6J origin and not an ES cell-derived one. By analyzing the karyotype of ES cell clones used in these experiments, it was found out that all three correctly targeted clones (E3B-589, E3B-849 and E3B-856) contained an abnormal chromosome number in the analyzed metaphases. This may explain the low chimerism observed in the G0 generation, and the fact that the targeted ES-derived Emilin3 allele was not germ-line transmitted to the F1 generation. The ES cell line (R1 line, passage p15) from which all three clones were derived was also checked cytogenetically. Interestingly, about 40% of analyzed metaphases were euploid cells, the rest of them contained either 39 or 41 chromosomes. Since it is known from the literature that ES cells with abnormal karyotypes are able to outgrow normal ones, especially with prolonged cell culture times, the heterogeneity of the ES cells is a likely explanation for the abnormal karyotypes detected in correctly targeted clones.In order to overcome this concern, for other ES cell lines that we have in our lab, karyotype analysis was performed. An ES cell line with the normal chromosome number will be used in order to inactivate Emilin3 gene using already prepared targeting vector (pKS-5.9DHM2neoTK).

Extracellular matrix; Emilin-3; gene targeting technology; Embryonic stem cells; chimeric mice

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