Characterization of clear. (a) Expression of csf1ra was analyzed by RT-PCR using cDNA from FACS-sorted populations of lymphocytes, progenitor cells, and myeloid cells obtained from the whole renal medulla (WKM) of a wild-type N. furzeri. Csf1ra was detected in all subpopulations, most strongly in myeloid cells. As a negative control, an RT sample (no reverse transcriptase during cDNA synthesis) was used to exclude genomic DNA contamination. As load control, rpl13a had been used. (B) Comparison of cell numbers in the different ERM subpopulations of fish with the following genotypes: mitzvah-/-ltk-/-csf1ra+/+ (n=2), mitzvah-/-ltk-/-csf1ra+/- (n=4), and mitzvah-/-ltk-/-csf1ra-/- (n=4). One-way ANOVA followed by Tukey’s post-hoc test revealed no significant differences. Horizontal line represents median. Whiskers show min to max value. (C, C’) Male clear fish (C) showed an appearance of melanophores on fin appendages, which was not observed in females (C’). (D,D’) Occurrence of melanophores intensified with age resulting in black fins in male fish (D). With woman clear animals (D’) black fins were not observed. Credit: eLife (2023). DOI: 10.7554/eLife.81549
The body pigmentation of an organism is caused by coloring substances and structures that are located, for example, in the cells of skin, hair, feathers or scales. This pigmentation considerably limits the investigation of important processes within a living organism (in vivo). To provide a detailed picture of the living body, scientists have used transparent model organisms.
Successfully generated transparent fish models, such as in zebrafish or medaka (Japanese ricefish), are already being used in cancer research. Lifespans of up to five years in zebrafish or two years in medaka have largely limited the use of those species in aging research, making such studies too lengthy and expensive.
Researchers at the Leibniz Institute on Aging – Fritz Lipmann Institute (FLI) in Jena have now succeeded in generating a transparent killifish (Nothobranchius furzeri), called klara, using CRISPR/Cas9 technology. With a lifespan of up to one year, klara facilitates in vivo studies of age-related processes. This means that new findings about the role and function of cells can be explored in much greater detail and detail in the future. The current study is now published in eLife.
Turquoise killifish – an animal model in aging research
“In recent years we have successfully introduced the turquoise killifish – which is native to East Africa and known in German as the turquoise beautiful ground carp for its beautiful, eye-catching color – as a new model organism in aging research at our institute.”, reports Prof. Christoph Englert, research group leader at the Leibniz Institute on Aging – Fritz Lipmann Institute (FLI) in Jena and professor of molecular genetics at FSU Jena.
“With a maximum lifespan of up to a year, this fish is so far the shortest-lived vertebrate that can be kept in the laboratory. In addition, it is genetically similar to humans, ages extremely quickly and shows typical signs of aging, making it very interesting for aging research,” adds Prof. Englert.
The full sequencing of the N. furzeri genome at the FLI has provided an important foundation for future analyses: the ability to specifically turn genes on and off, thus learning about the influence of individual genes on aging or in relation to aging – related diseases.
Pigmentation inactivation using CRISPR/Cas9
“In the turquoise killifish, there are three types of pigment cells that are responsible for the very beautiful coloration of the fish,” explains Dr. Johannes Krug, a postdoc in the Englert Research Group.
“The availability of the killifish genome sequence gave us the opportunity to investigate whether sequence-specific genome editing methods, such as CRISPR/Cas9, can be used to inactivate genes responsible for body pigmentation to obtain a transparent fish for use in aging research. These studies were the main focus of my dissertation at FLI.”
‘Klara’ – the transparent killifish
CRISPR/Cas9 is a molecular biology method that can be used to inactivate or modify genes in a sequence-specific manner, as if they were cut with scissors. By applying this method, the Jena researchers succeeded in deactivating the genes responsible for the fish’s pigmentation, creating a transparent killifish for the first time. The transparent fishing line, which the researchers named klara (“klar” is the German word for “clear”), now gives researchers a clear view of the internal organs and their development in a living animal.
The transparent fishing line currently includes about 200 animals at the FLI, both males and females, which can now be used in numerous aging studies.
What role do senescent cells play in the aging process?
“Our transparent fish has great potential for aging research and opens up a wide range of completely new applications. In my group, the new transparent fish line is already being used for in vivo studies of senescent cells,” reports Prof. Englert. Senescent cells are cells that have stopped dividing and attack surrounding cells and tissues by continuously releasing pro-inflammatory factors. Until now, little is known about the role and function of these cells.
It is known that in humans, but also in mice or killifish, the number of senescent cells increases with age, creating a kind of permanent inflammation in the body during the aging process. The targeted removal of senescent cells can therefore contribute to better health or even slow down the aging process. The research and development of substances known as senolytics, which can effectively remove senescent cells from the body, is therefore of great importance not only to the pharmaceutical and anti-aging industry, but also to the research of aging.
“With the Klara line, we can now investigate the role of senescent cells in the living organism at the molecular level. By labeling them with fluorophores and then examining them under a fluorescence microscope, we can learn where they appear in the body and whether they may be clustered in certain locations, and what effects their removal has on surrounding cells and tissues,” says Dr. Krug, highlighting the benefits of Klara. This will lead to new insights into the role and function of this specific cell population during aging.
Johannes Krug et al, Generation of a transparent killifish line through multiplex CRISPR/Cas9-mediated gene silencing, eLife (2023). DOI: 10.7554/eLife.81549
Offered by the Fritz Lipmann Institute
Quote: Klara, a transparent fish for aging research (2023, March 17) Retrieved March 19, 2023 from https://phys.org/news/2023-03-klara-transparent-fish-aging.html
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