diplomsko delo
Iva Matić (Author), Tadeja Režen (Mentor)

Abstract

Krožne RNA uravnavajo številne procese v celicah in sodelujejo pri razvoju bolezni. Mnoge imajo spremenjeno izražanje v rakavih celicah in njihovo vlogo v razvoju raka, zlasti hepatocelularnega karcinoma (HCC), preučujemo v celičnih modelih. Namen diplomske naloge je bil priprava lentivirusnih vektorjev za prekomerno in utišano izražanje krožnih RNA. Kot kandidata za kloniranje vektorjev smo izbrali eksosomski krožni RNA hsa_circ_0001861 in hsa_circ_0070039, ki sta v raku jeter znižani. Utišanega izražanja nismo mogli določati, saj smo ugotovili, da se siRNA vežejo tudi na druge tarčne mRNA. Posledično bi bili zaradi prevelike nespecifičnosti utišani tudi drugi geni. Z metodami molekulskega kloniranja kot so PCR pomnoževanje, restrikcija, ligacija in transformacija, nam je uspelo vstaviti željeno zaporedje hsa_circ_0001861 v plazmidni vektor pRRcirc, kar smo tudi dokazali z določanjem nukleotidnega zaporedja z metodo po Sangerju. Ker imajo circRNA zelo kratko zaporedje, ki je specifično le za njih, je njihova detekcija, pomnoževanje in utišanje pogosto omejeno, kar onemogoča raziskave njihove funkcije v celičnih linijah.

Keywords

rak jeter;hepatocelularni karcinom;krožne RNA;circRNA;hsa_circ_0001861;hsa_circ_0070039;utišanje genov;lentivirusni vektorski sistem;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [I. Matić]
UDC: 577.21:616.36(043.2)
COBISS: 126640899 Link will open in a new window
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Downloads: 10
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Other data

Secondary language: English
Secondary title: Preparation of lentiviral vectors for regulation of circRNA expression in cell lines
Secondary abstract: Circular RNAs regulate many processes in cells and participate in the development of diseases. Many have altered expression in cancer cells and their role in cancer development, especially hepatocellular carcinoma (HCC), is studied in cell models. The aim of the work was the preparation of lentiviral vectors for the overexpression and silenced expression of circular RNAs. As candidates for vector cloning, we selected exosomal circular RNAs hsa_circ_0001861 and hsa_circ_0070039, which are downregulated in liver cancer. We could not determine silenced expression, as we found that siRNAs also bind to other target mRNAs. As a result, other genes would also be silenced due to excessive non-specificity. Using molecular cloning methods such as PCR amplification, restriction, ligation and transformation, we managed to insert the desired sequence hsa_circ_0001861 into the plasmid vector pRRcirc, which we also proved by determining the nucleotide sequence by Sanger method. Because circRNAs have a very short sequence specific to them, their detection, amplification and silencing are often limited, which makes research of their function in cell lines impossible.
Secondary keywords: circular RNA;liver cancer;hsa_circ_0001861;hsa_circ_0070039;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 45 str.
ID: 16391450