Izražaj gena Stam2 određen na miševima preinačenim genskom zamkom

Ćurlin, Marija (2006) Izražaj gena Stam2 određen na miševima preinačenim genskom zamkom. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Signal transduction and receptor downregulation by endocytic pathway are crucial processes in mammalian embryonic development and normal functioning of nervous system. STAM2 is a protein located on early endosomes and evidences exist that it participates in receptor sorting during endocytic pathway. The aim of the research was to determine Stam2 expression pattern in mouse embryos and the brain of adult Stam2Gt1Gaj mice by histochemical staining for ß-galactosidase in order to get insight into its possible role in the development of the mouse embryos and adult central nervous system. The mouse line Stam2Gt1Gaj was produced by gene trap method where the pKC199ßgeo vector was introduced into Stam2 gene, and consequently impaired the STAM2 protein production and functioning. The gene trap vector contained promoterless lacZ gene coding for ß-galactosidase; hence the ß-galactosidase activity reflected the Stam2 expression. Analysis of the modified Stam2 gene and its transcripts and STAM2 protein revealed a mutant transcript giving a fusion protein with ß-galactosidase activity. Additionally, an alternative splicing event was found to occur during Stam2 transcription, but it did not result in STAM2 protein. The normal Stam2 transcript was present in all adult wild type mice organs tested. Stam2 expression detected by histochemical staining for ß-galactosidase was present in mouse embryos between developmental stages E9.5 and E18.5 in primitive hindgut, future central nervous system and heart, and toward the end of gestation in some organs of endocrine system and in the covering epithelia. Stam2 was strongly expressed in the forebrain cortex, hippocampus and olfactory bulbs of newborn as well as adult mice. It was also visible in the grey matter nuclei and the cerebellar cortex. The results of this research showed a possible role of STAM2 in the development of nervous system, heart, endocrine glands and epithelial tissues and indicated that could be related to its role in signal transduction regulation by endocytic pathway.

Abstract in Croatian

Prijenos podražaja i njegova regulacija pomoću endocitoze i razgradnje receptora u lizosomima važni su procesi u embrionalnom razvoju sisavaca, te u razvoju i normalnom funkcioniranju živčanog sustava. STAM2 je bjelančevina smještena na ranim endosomima, te postoje dokazi da sudjeluje u razvrstavanju receptora namijenjenih razgradnji u lizosomima. Cilj rada bio je odrediti područja izražaja gena Stam2 praćenjem aktivnosti enzima ß-galaktozidaze na zamecima i mozgovima odraslih miševa linije Stam2Gt1Gaj i time dobiti nove spoznaje o njegovoj mogućoj ulozi u razvoju mišjih zametaka i funkcioniranju središnjeg živčanog sustava odraslih miševa. Mišja linija Stam2Gt1Gaj dobivena je postupkom genske zamke gdje vektor pKC199ßgeo ugradnjom u gen Stam2 onemogućuje stvaranje, a time i funkcioniranje normalne bjelančevine STAM2. Vektor genske zamke sadrži gen za ß-galaktozidazu bez vlastitog promotora, pa se praćenjem aktivnosti ß-galaktozidaze može odrediti izražaj gena Stam2. Analiza preinačenog gena Stam2, te njegovih transkripata i bjelančevine STAM2, pokazala je da preinačeni gen daje promijenjeni transkript od kojeg nastaje stopljena bjelančevina s aktivnošću ß-galaktozidaze, te da se tijekom transkripcije događa alternativno prekrajanje gena Stam2, koje ne uzrokuje stvaranje bjelančevine STAM2. Normalni transkript gena Stam2 prisutan je u svim ispitivanim organima odraslog miša divljeg tipa. Izražaj Stam2 određen histokemijskim bojanjem na ß-galaktozidazu vidljiv je između razvojnih stadija E9,5 i E18,5 u primitivnom stražnjem crijevu, osnovi središnjeg živčanog sustava i srcu, te pred kraj gestacijskog razdoblja u organima endokrinog sustava i pokrovnom epitelu zametaka. U mozgu novorođenih kao i odraslih miševa Stam2 je intenzivno izražen na području kore velikog mozga, hipokampusa i olfaktornih bulbusa, a vidljiv je i u jezgrama sive tvari velikog mozga, te kori malog mozga. Rezultati ovog istraživanja ukazuju na moguću ulogu STAM2 u razvoju živčanog sustava, srca, endokrinih žlijezda i epitelnih tkiva, te da bi ta uloga mogla biti povezana s ulogom STAM2 u regulaciji prijenosa podražaja pomoću endocitoze.

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