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Ugotavljanje morebitne patogenosti mutacij gena ATG16L1 z uporabo humanizirane kvasovke
Posinek, Matevž (Author), Petrovič, Uroš (Mentor) More about this mentor... This link opens in a new window

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Abstract
Metoda z uporabo humanizirane kvasovke temelji na vstavitvi človeškega gena v sev kvasovke, ki ima izbit homolog človeškega gena, ki ga želimo testirati. Vstavljen človeški gen naj bi nadomestil funkcijo izbitega gena kvasovke, učinkovitost komplementacije pa lahko zaznamo s spremembo fenotipa kvasovke. Za nadomestitev lahko uporabimo tudi mutirane alele človeških genov in na osnovi sposobnosti komplementacije s strani mutiranih alelov napovedujemo, ali imajo mutacije negativen vpliv na normalno funkcijo gena. V sklopu naše raziskave smo želeli ugotoviti, ali lahko človeški gen ATG16L1 uspešno nadomesti funkcijo kvasovkinega homolognega gena ATG16. Gena sta udeležena v procesu avtofagije, mutacije v genu ATG16L1 pa so povezane s tveganjem za razvoj Chronove bolezni. V primeru uspešne komplementacije je bil naš naslednji cilj ugotoviti, če sta mutaciji 220G>A (Asp74Asn) in 898A>G (Thr300Ala) gena ATG16L1 škodljivi oziroma patogeni. Zamenjavo genov smo v delecijski mutanti laboratorijskega seva BY4741 atg16? izvedli s tehniko za urejanje genoma CRISPR/Cas9. Humaniziran sev smo nato podvgrli seriji fenotipskih testov in na osnovi rezultatov ugotovili, da gen ATG16L1 ni sposoben nadomestiti funkcije kvasovkinega gena ATG16. Nadaljnje ugotavljanje škodljivosti mutacij gena ATG16L1 z uporabo humanizirane kvasovke posledično ni bilo mogoče.

Language:Slovenian
Keywords:humanizirana kvasovka, CRISPR/Cas9, alel, avtofagija
Work type:Master's thesis/paper (mb22)
Organization:BF - Biotechnical Faculty
Year:2019
COBISS.SI-ID:5329231 Link is opened in a new window
Views:280
Downloads:118
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Secondary language

Language:English
Title:Identifying potential pathogenicity of mutations in gene ATG16L1 with the use of humanized yeast
Abstract:
The method using humanized yeast assumes a substitution of a gene in yeast with a homologous human gene under study. The inserted human gene is supposed to complement the function of the yeast gene. This can be detected as a change in yeast strain's phenotype. Different variants of human gene can then be used for the substitution. Based on their ability to complement the yeast counterpart, we can asses whether these variants have a negative effect on the normal function of a gene. We wanted to test the capability of human gene ATG16L1 to complement the homologous yeast gene ATG16. Both genes are involved in the proces of autophagy, and mutations in gene ATG16L1 have been associated with an increased risk of Chron's disease. In case of successful complementation, our next goal was to asses whether mutations 220G>A (Asp74Asn) and 898A>G (Thr300Ala) in gene ATG16L1 are pathogenic. With the use of genome editing technique CRISPR/Cas9, we performed a gene substitution in the yeast deletion strain atg16Δ. We then performed a series of phenotypic tests on the humanized strain, which showed that ATG16L1 is not capable of complementing the ATG16 gene. Further identification of pathogenicity for different variants of gene ATG16L1 was therefor impossible.

Keywords:humanized yeast, CRISPR/Cas9, gene variants, autophagy

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