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Making Transgenic \kur{C. elegans} with Polycistronic mCherry Vector
FARKA, Dominik
Creation of transgenic animals has become a popular method to analyse gene function. In the nematode Ceanorhabditis elegans transformation is widely used and can be achieved by microinjection. For functional analyses, transgene constructs typically contain a promoter driving the expression of the protein of interest that is fused to a fluorescent protein. However, as this fusion of proteins can lead to misfolding of the protein of interest and may not reflect proper function, a modification of the expression vector has been developed; introducing a short sequence of non-coding DNA in-between the sequences of the two proteins and making the construct compatible with a polycistronic operon system. In this study, four different polycistronic constructs were introduced into C. elegans by means of microinjection in order to provide new tools for the analyses of gene function. Tissue specific promoters wrt-2 (seam cells), grl-21 (hyp7), and egl-17 (vulval precursor cells) were used to over-express either NHR-25 or SMO-1 in the corresponding tissues and the expression was visualized by independently translated mCherry red fluorescent. 10 independent transformed C. elegans strains were established and corresponding tissue-specific promoter activities were confirmed. Furthermore, in some cases, ectopic behaviour was observed e.g. ectopic mCherry expression in different tissues or specific cell differentiation defects that was most likely caused by the overexpression of NHR-25 or SMO-1. This study was the first case in our laboratory to generate transformed C. elegans utilizing the polycistronic mCherry vector system. New genetic tools were introduced in the laboratory useful for further analyses of gene function.
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The transcriptional regulation by the nuclear receptor NHR-25 in \kur{Caenorhabditis elegans}
MERGLOVÁ, Linda
NHR-25 je jedním z mála konzervovaných jaderných receptorů haďátka C. elegans a jeho rodina je zahrnuta v mnoha vývojových procesech nejen u háďátka, ale také u octomilek, ryb, myší a člověka. Buněčný mechanismus funkce této genové rodiny je však zatím málo znám. U C. elegans pravděpodobně funguje jako transkripční faktor, ale jeho přímý cílový gen doposud nebyl identifikován. V této studii jsem použila definovaná vazebná místa pro NHR-25 a GFP jako marker pro zviditelnění možnosti, že NHR-25 reguluje transkripci jiného gebu (genů) in vivo. Vyzkoušela jsem také alternativní přístup se dvěma geny v již existujících transgenních kmenech háďátek, nesoucích promotor::GFP fúzované transgeny exprimované v epiteliálních buňkách, mohou-li být tyto geny regulované NHR-25. Výsledky naznačují, že jeden ze zkoumaných genů může být ve vztahu k nhr-25 a tento receptor může být i aktivním při endocytóze.
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