Priprava humaniziranih sevov kvasovke za funkcijsko analizo patogenih variant genov ING1 in ING3

Šalamon, Iris (author), Petrovič, Uroš (mentor)

Kvasovka Saccharomyces cerevisiae je odličen modelni organizem za preučevanje človeških homolognih genov. Dostopnost metod za natančno spreminjanje dednine in ohranjenost genov nam omogočata zamenjavo posameznih genov ali celotnih metabolnih poti. Z zamenjavo lahko predvidimo potencialni vpliv posameznih različic preučevanega gena na kvasovko oziroma na človeka. Takšno orodje bi nam omogočilo hitro in učinkovito testiranje v zelo kratkem času. Cilj naše naloge je bil ugotoviti, ali lahko gena ING1 oziroma ING3 nadomestita kvasni gen PHO23 ter v nadaljevanju testirati vpliv različic genov na fenotip kvasovke. Z metodo CRISPR-Cas9 smo na izbrano mesto uspešno vstavili oba človeška gena. Izbrana metoda je hitra in v kvasovki zelo učinkovita. Metode, s katerimi preverjamo razliko med divjim tipom kvasovke in izbranim mutantom, so enostavne. Testirali smo tri različne fenotipske teste, od katerih je bil ustrezen eden. Dodatek CaCl2 v gojišču inhibira rast delecijskega seva, rast divjega tipa pa je nekoliko zmanjšana. Po uspešni vstavitvi človeških genov v kvasovko smo s fenotipskimi testi ugotovili, da imata humanizirani kvasovki z genom ING1 oziroma z genom ING3 enak fenotip kot delecijski sev pho23?. Posameznih različic gena ING1 oziroma ING3 zato nismo testirali. Na podlagi izolirane RNA sklepamo, da se človeški gen ING1 v kvasovki prepisuje. Tega ne moremo sklepati za človeški gen ING3.

• Language: Slovenian
• Keywords: kvasovke, humanizirane kvasovke, CRISPR-Cas9, PHO23, ING1, ING3
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-110018
• COBISS.SI-ID: 5331279
• Publication date in RUL: 11.09.2019

Secondary language

• Language: English
• Title: Development of humanized yeast strains for functional analysis of pathogenic variants of ING1 and ING3 genes

Abstract

Yeast Saccharomyces cerevisiae is a useful model organism which can be easily genetically manipulated. High degree of conservation between the yeast and human homologous genes enables us to swap genes or entire pathways. This let us study the effect of human gene variants in yeast. The aim of this thesis was to find out if human genes ING1 and ING3 can substitute yeast gene PHO23. Furthermore, we wanted to test how different variants of the human genes affect the ability of the human protein to complement the yeast mutant. The substitution was made by a quick and effective method CRISPR-Cas9. We successfully swapped either of the selected human genes into the desired genome locus. We tested different phenotypes to find one that can distinguish between the deletion mutant strain and the wild type strain. From the three different phenotypes that we tested only one proved suitable. The CaCl2 sensitivity was tested in the mutant and the wild type strains. The growth of the deletion mutant strain was reduced in comparison to the wild type. We found out that strains carrying either human gene ING1 or ING3 exhibit the same phenotype as the pho23? deletion strain. The effect of different ING1 and ING3 variants therefore was not tested. Based on subsequent RNA isolation we propose that human gene ING1 is transcribed in yeast but its product cannot complement yeast gene PHO23 product.

• Keywords: yeast, humanized yeast, CRISPR-Cas9, PHO23, ING1, ING3