Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies
Gene expression in eukaryotes are regulated through complex interactions between regulatory proteins, cis-regulatory sequences as well as chemical modifications and structure of chromatin. The function of cis-regulatory regions can be studied through expression of reporter genes in cell cultures. Id...
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Norwegian University of Life Sciences, Ås
2020
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Online Access: | https://hdl.handle.net/11250/2725959 |
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ftunivmob:oai:nmbu.brage.unit.no:11250/2725959 2023-05-15T15:26:17+02:00 Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies Optimalisering av transfeksjon av primære hepatocytter fra atlanterhavslaks for funksjonelle studier Wilberg, Ragnhild Sandve, Simen Rød 2020 application/pdf https://hdl.handle.net/11250/2725959 eng eng Norwegian University of Life Sciences, Ås https://hdl.handle.net/11250/2725959 Attribution-NonCommercial-NoDerivatives 4.0 Internasjonal http://creativecommons.org/licenses/by-nc-nd/4.0/deed.no CC-BY-NC-ND Master thesis 2020 ftunivmob 2021-09-23T20:15:39Z Gene expression in eukaryotes are regulated through complex interactions between regulatory proteins, cis-regulatory sequences as well as chemical modifications and structure of chromatin. The function of cis-regulatory regions can be studied through expression of reporter genes in cell cultures. Ideally such reporter gene expression should be studied in biologically relevant cells, like primary cell cultures. The primary aim of this work was to optimize a protocol for transfection of primary hepatocytes from Atlantic salmon, to be able to perform functional studies on gene expression in liver. The secondary aim was to test this protocol by performing a promoter reporter assay on the cis-regulatory elements driving the expression in two duplicated Atlantic salmon fatty acyl elongase 5 genes involved in the endogenous synthesis of long-chain polyunsaturated fatty acid (LC-PUFA) synthesis in liver. Transfection optimizations were done using both cationic lipid-mediated transfection and electroporation. Results from these optimization experiments suggests that electroporation is the superior option for transfecting primary Atlantic salmon hepatocytes, with transfection efficiency up to 30%. We also found that perfusion is an important, but technically challenging element for successful transfection in these cells. Finally, our experiment with the elovl5 promoter sequences showed induced reporter expression in hepatocytes. In conclusion, we have developed a robust transfection protocol which paving the way for functional studies of gene regulatory logics in liver cells. Genuttrykk i eukaryoter er regulert gjennom komplekse interaksjoner mellom regulatoriske proteiner, cisregulatoriske sekvenser og kjemiske og strukturelle endringer av kromatin. Funksjonen av cisregulatoriske sekvenser kan studeres gjennom uttrykk av reportergener i cellekulturer. Ideelt sett bør slike reportergenstudier utføres i biologisk relevante celler, som for eksempel primæcellekulturer. Hovedmålet med dette arbeidet var derfor å optimalisere transfeksjon av primære hepatocytter fra atlanterhavslaks, for å kunne utføre funksjonelle studier av genuttrykk i lever. Det sekundære målet var å teste denne protokollen ved å sammenlikne cis-regulatoriske sekvenser fra to elovl5-genduplikater i atlanterhavslaks. Disse to genene koder for elovl5-enzymer som er involvert i den endogene syntesen av langkjedede flerumettede fettsyrer i lever. Transfeksjonsoptimaliseringen ble gjort både for kationisk lipid-mediert tranfeksjon og elektroporering. Resultatene fra optimaliseringsforsøkene viste at elektroporering er det mest effektive alternativet for å transfektere primære hepatocytter fra atlanterhavslaks, med en transfeksjonseffiktivitet på 30%. I tillegg fant vi at perfusjon er et viktig, men teknisk utfordrende element for vellykket transfeksjon. Til sist viste vi at elovl5-promotere kan indusere transkripsjon i primære leverceller. Vi konkluderer med at vår nye transfeksjonsprotokoll vil kunne være et viktig verktøy for fremtidige studier av genregulering i leverceller. Norges forskningsråd: FRIMEDBIO Småforsk Biovit 2019 M-BIOTEK Master Thesis Atlanterhavslaks Atlantic salmon Open archive Norwegian University of Life Sciences: Brage NMBU Lever ENVELOPE(-63.608,-63.608,-65.506,-65.506) |
institution |
Open Polar |
collection |
Open archive Norwegian University of Life Sciences: Brage NMBU |
op_collection_id |
ftunivmob |
language |
English |
description |
Gene expression in eukaryotes are regulated through complex interactions between regulatory proteins, cis-regulatory sequences as well as chemical modifications and structure of chromatin. The function of cis-regulatory regions can be studied through expression of reporter genes in cell cultures. Ideally such reporter gene expression should be studied in biologically relevant cells, like primary cell cultures. The primary aim of this work was to optimize a protocol for transfection of primary hepatocytes from Atlantic salmon, to be able to perform functional studies on gene expression in liver. The secondary aim was to test this protocol by performing a promoter reporter assay on the cis-regulatory elements driving the expression in two duplicated Atlantic salmon fatty acyl elongase 5 genes involved in the endogenous synthesis of long-chain polyunsaturated fatty acid (LC-PUFA) synthesis in liver. Transfection optimizations were done using both cationic lipid-mediated transfection and electroporation. Results from these optimization experiments suggests that electroporation is the superior option for transfecting primary Atlantic salmon hepatocytes, with transfection efficiency up to 30%. We also found that perfusion is an important, but technically challenging element for successful transfection in these cells. Finally, our experiment with the elovl5 promoter sequences showed induced reporter expression in hepatocytes. In conclusion, we have developed a robust transfection protocol which paving the way for functional studies of gene regulatory logics in liver cells. Genuttrykk i eukaryoter er regulert gjennom komplekse interaksjoner mellom regulatoriske proteiner, cisregulatoriske sekvenser og kjemiske og strukturelle endringer av kromatin. Funksjonen av cisregulatoriske sekvenser kan studeres gjennom uttrykk av reportergener i cellekulturer. Ideelt sett bør slike reportergenstudier utføres i biologisk relevante celler, som for eksempel primæcellekulturer. Hovedmålet med dette arbeidet var derfor å optimalisere transfeksjon av primære hepatocytter fra atlanterhavslaks, for å kunne utføre funksjonelle studier av genuttrykk i lever. Det sekundære målet var å teste denne protokollen ved å sammenlikne cis-regulatoriske sekvenser fra to elovl5-genduplikater i atlanterhavslaks. Disse to genene koder for elovl5-enzymer som er involvert i den endogene syntesen av langkjedede flerumettede fettsyrer i lever. Transfeksjonsoptimaliseringen ble gjort både for kationisk lipid-mediert tranfeksjon og elektroporering. Resultatene fra optimaliseringsforsøkene viste at elektroporering er det mest effektive alternativet for å transfektere primære hepatocytter fra atlanterhavslaks, med en transfeksjonseffiktivitet på 30%. I tillegg fant vi at perfusjon er et viktig, men teknisk utfordrende element for vellykket transfeksjon. Til sist viste vi at elovl5-promotere kan indusere transkripsjon i primære leverceller. Vi konkluderer med at vår nye transfeksjonsprotokoll vil kunne være et viktig verktøy for fremtidige studier av genregulering i leverceller. Norges forskningsråd: FRIMEDBIO Småforsk Biovit 2019 M-BIOTEK |
author2 |
Sandve, Simen Rød |
format |
Master Thesis |
author |
Wilberg, Ragnhild |
spellingShingle |
Wilberg, Ragnhild Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies |
author_facet |
Wilberg, Ragnhild |
author_sort |
Wilberg, Ragnhild |
title |
Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies |
title_short |
Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies |
title_full |
Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies |
title_fullStr |
Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies |
title_full_unstemmed |
Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies |
title_sort |
optimization of transfection of primary hepatocytes from atlantic salmon for functional studies |
publisher |
Norwegian University of Life Sciences, Ås |
publishDate |
2020 |
url |
https://hdl.handle.net/11250/2725959 |
long_lat |
ENVELOPE(-63.608,-63.608,-65.506,-65.506) |
geographic |
Lever |
geographic_facet |
Lever |
genre |
Atlanterhavslaks Atlantic salmon |
genre_facet |
Atlanterhavslaks Atlantic salmon |
op_relation |
https://hdl.handle.net/11250/2725959 |
op_rights |
Attribution-NonCommercial-NoDerivatives 4.0 Internasjonal http://creativecommons.org/licenses/by-nc-nd/4.0/deed.no |
op_rightsnorm |
CC-BY-NC-ND |
_version_ |
1766356792690868224 |