Evaluation of mRNA translation capacity in zebrafish model
Arola, Henri (2022)
2022
Lääketieteen lisensiaatin tutkinto-ohjelma - Licentiate's Programme in Medicine
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2022-05-02
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202204153252
https://urn.fi/URN:NBN:fi:tuni-202204153252
Tiivistelmä
The first commercially available messenger ribonucleic acid (mRNA) vaccines were developed against COVID- 19 disease causing corona virus, but the same technique is also applicable to other pathogens. An important aspect that needs to be considered in the vaccine development is the translation capacity of the mRNA molecule which is essential for the efficiency of the vaccine. One way to increase the translation capacity is to modify the uridine base in the mRNA. The development of a new vaccine is a time-consuming process. In vitro data is in important role in vaccine development and different animal models have proved to be invaluable in the development process. Zebrafish are well suited for immunological studies as the immune system of the fish is fairly similar to human immune system. The properties of zebrafish make them a fascinating option for vaccine studies.
In this work the translation capacity of mRNA molecules constructed with regular uridine base (UTP) or 1-methyl-pseudo-uridine (p-UTP) is evaluated in zebrafish model. The translation capacities of the two different mRNA molecules are compared between each other and to pcDNA3.1-egfp plasmid that was used to construct the mRNA molecules. To assess the mRNA and pcDNA3.1-egfp plasmid expression a gene coding for an enhanced green fluorescent protein (egfp) was used which allowed the evaluation of mRNA expression through fluorescence of the developing larvae. The mRNA or pcDNA3.1-egfp plasmid were delivered to the larvae by microinjection to zebrafish embryos.
The developing larvae were followed for 120-hour period, and they were imaged with fluorescence microscope at regular 24-hour intervals. To separate real fluorescence due to expression of egfp from the auto fluorescence of zebrafish larvae, uninjected and water injected larvae were also included in the study. The assessment of fluorescence was made with naked eye three times for each fluorescent image on a three-step evaluation scale. Water injected larvae were also used to evaluate the effect of microinjection for the survival of the developing larvae as they provided a way to separate the effect of microinjection from the effect of injected substance.
Against the expectations, the fluorescence was stronger in the larvae injected with regular mRNA and only a weak fluorescence could be seen in the larvae injected with p-UTP modified mRNA. The fluorescence in larvae injected with pcDNA3.1-egfp plasmid was comparable to the fluorescence of larvae injected with regular mRNA. The decrease in survival of the larvae from microinjection was small as approximately 10 per cent of the uninjected larvae died during the follow-up period compared to approximately 12 per cent of the larvae injected with water. The mortality was doubled compared to uninjected larvae for both the mRNA injected groups and was approximately 20 per cent during the follow-up period. Injection with pcDNA3.1-egfp was clearly harmful for the developing larvae as approximately 50 per cent of them died during the research.
In this work the translation capacity of mRNA molecules constructed with regular uridine base (UTP) or 1-methyl-pseudo-uridine (p-UTP) is evaluated in zebrafish model. The translation capacities of the two different mRNA molecules are compared between each other and to pcDNA3.1-egfp plasmid that was used to construct the mRNA molecules. To assess the mRNA and pcDNA3.1-egfp plasmid expression a gene coding for an enhanced green fluorescent protein (egfp) was used which allowed the evaluation of mRNA expression through fluorescence of the developing larvae. The mRNA or pcDNA3.1-egfp plasmid were delivered to the larvae by microinjection to zebrafish embryos.
The developing larvae were followed for 120-hour period, and they were imaged with fluorescence microscope at regular 24-hour intervals. To separate real fluorescence due to expression of egfp from the auto fluorescence of zebrafish larvae, uninjected and water injected larvae were also included in the study. The assessment of fluorescence was made with naked eye three times for each fluorescent image on a three-step evaluation scale. Water injected larvae were also used to evaluate the effect of microinjection for the survival of the developing larvae as they provided a way to separate the effect of microinjection from the effect of injected substance.
Against the expectations, the fluorescence was stronger in the larvae injected with regular mRNA and only a weak fluorescence could be seen in the larvae injected with p-UTP modified mRNA. The fluorescence in larvae injected with pcDNA3.1-egfp plasmid was comparable to the fluorescence of larvae injected with regular mRNA. The decrease in survival of the larvae from microinjection was small as approximately 10 per cent of the uninjected larvae died during the follow-up period compared to approximately 12 per cent of the larvae injected with water. The mortality was doubled compared to uninjected larvae for both the mRNA injected groups and was approximately 20 per cent during the follow-up period. Injection with pcDNA3.1-egfp was clearly harmful for the developing larvae as approximately 50 per cent of them died during the research.