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Priprava lentivirusnih vektorjev za regulacijo izražanja circRNA v celičnih linijah
ID Matić, Iva (Author), ID Režen, Tadeja (Mentor) More about this mentor... This link opens in a new window

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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.

Language:Slovenian
Keywords:krožne RNA, rak jeter, hsa_circ_0001861, hsa_circ_0070039
Work type:Bachelor thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2022
PID:20.500.12556/RUL-139729 This link opens in a new window
COBISS.SI-ID:126640899 This link opens in a new window
Publication date in RUL:06.09.2022
Views:629
Downloads:33
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Secondary language

Language:English
Title:Preparation of lentiviral vectors for regulation of circRNA expression in cell lines
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.

Keywords:circular RNA, liver cancer, hsa_circ_0001861, hsa_circ_0070039

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