engleski

Microscopy in expression pattern determination and functional analysis of signal transducing adaptor molecule 2 (Stam2)

STAM2 (Signal transducing adaptor molecule 2) is a signaling adaptor presumed to act in the intracellular cargo sorting for degradation at the level of the late endosome (1). Although its function was already assessed at the cellular level in permanent cell cultures, its function in mammalian organism could be revealed only through genetic modification at the level of the whole animal. In order to characterize Stam2 function in mouse, the gene trap method was applied. It comprises the genetic modification of embryonic stem cells with a non-homologous vector containing a splice acceptor and fused lacZ and neoR genes without their own promoters. In case the vector was inserted in an endogenous gene, the splice acceptor would be recognized and lacZ and neoR genes would be driven by the promoter of the endogenous gene. This enabled to screen the genetically modified embryonic stem cells and the corresponding mouse lines for lacZ activity, which mirrored the expression of the endogenous gene. In addition the vector insertion disturbed the proper protein translation 3’ to the gene trap vector. After the selection process, the mouse line carrying the gene trap mutation in Stam2 gene was chosen for further characterization. The molecular analysis defined the exact place of the insertion and its consequences on the mRNA and the protein level. This showed that the gene trap vector was between exon 2 and 3, and the STAM2 protein production in the homozygous mice was highly diminished (2). Stam2 expression pattern was assessed by monitoring lacZ activity in the investigated tissues. lacZ gives rise to beta-galactosidase, which changes the color of its substrate X-gal in blue. Stam2 expression started in 8.5-day embryos in the hind gut. In 9.5-day embryos it was present in the heart, while in 10.5-day embryos appeared in the ventral part of the neural tube (Figure 1). The expression in the heart persisted during embryo development and continued in the adults. The expression in the neural tube was continuous to the expression in the brain, in particular from 16.5-day embryos in the brain cortex, and from 18.5-day embryos in the hippocampus. In addition to the brain and the heart, Stam2 was expressed as well in the testis and developing adrenal and pituitary glands. In order to assess Stam2 function the phenotype of the homozygous carriers of gene trap mutation was compared with the wild type mice. The homozygotes were alive, fertile and did not show any major abnormalities. The functional studies of mouse behavior were combined with detailed morphological analysis of embryos and adult organs and tissues. The morphological studies involved the classical histology using the hematoxylin-eosin staining, the semithin sections stained by toluidine blue, immunohistochemistry of the selected markers, and finally the ultrastructural analysis using electron microscope (Figure 2). These studies are expected to reveal the fine differences in phenotypes of the wild type and the mutant mice, which would then reveal the Stam2 function in mouse, in particular in the mouse brain.

Stam2; brain; mouse

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