Characterization of Clara. (ONE) Expression of csf1ra analyzed by RT-PCR using cDNA from FACS-sorted populations of lymphocytes, progenitor cells and myeloid cells obtained from wild-type whole kidney medulla (WKM) N. furzeri. Csf1ra was detected in all subpopulations, most strongly in myeloid cells. As a negative control, an RT sample (without reverse transcriptase during cDNA synthesis) was used to exclude genomic DNA contamination. As a load control, rpl13a was used. (si) Comparison of cell numbers in the different subpopulations of WKM fish with the following genotypes: mitfa–/-ltk–/-csf1ra+/+ (n=2), mitfa–/-ltk–/-csf1ra+/- (n=4), and mitfa–/-ltk–/-csf1ra–/- (n=4). One-way ANOVA followed by Tukey’s post hoc test revealed no significant differences. The horizontal line represents the median. Whiskers show min. to the max. value. (C, C’) Male Clara fish (do) showed appearance of melanophores on wing appendages, which was not observed in females (DO’). (D, D’) The appearance of melanophores intensified with age resulting in black fins in male fish (Hey). In female Clara animals (HEY’) no black fins were observed. Credit: eLife (2023). DOI: 10.7554/eLife.81549
The pigmentation of an organism’s body is caused by pigments and structures found in the cells of the skin, hair, feathers or scales, for example. This pigmentation significantly limits investigations of important processes within a living organism (in vivo). To provide a detailed view of the living body, scientists used transparent model organisms.
Transparent fish models that have been successfully created, such as in zebrafish or medaka (Japanese rice fish), are already being used in cancer research. Lifespans of up to five years in the zebra or two years in the medaka have greatly limited the use of these species in aging research, making such research too time-consuming and expensive.
Researchers at the Leibniz Institute for Aging — Fritz Lipmann Institute (FLI) in Jena have now succeeded in creating a transparent deadly fish (Nothobranchius furzeri), called klara, using CRISPR/Cas9 technology. With a shelf life of only one year at most, klara facilitates in vivo studies of age-related processes. This means that new findings about the role and function of cells can be explored much more fully and in greater detail in the future. The current study has now been published in eLife.
Turquoise killifish – an animal model in aging research
“In recent years, we have successfully established the turquoise killifish – originally from East Africa and known in German as the turquoise magnificent carp, due to its beautiful, striking coloration – as a new model organism in aging research at the institute us,” reports Prof. Christoph Englert, head of the research group at the Leibniz Institute for Aging—Fritz Lipmann Institute (FLI) in Jena and Professor of Molecular Genetics at FSU Jena.
“With a maximum lifespan of up to one 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, which makes it very interesting for aging research,” adds Professor Englert.
By sequencing the complete genome of N. furzeri at FLI, an important basis for future analyzes has become: the ability to specifically turn genes on and off and thus learn about the effect of individual genes on or in relation to senescence- related diseases.
Disabling pigmentation using CRISPR/Cas9
“In the turquoise killifish, there are three types of pigment cells that are responsible for the very beautiful coloring of the fish,” explains Dr. Johannes Krug, a postdoctoral researcher 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 could be used to turn off the genes responsible for body pigmentation to obtain a transparent fish for use in aging research. These studies were the main objective of my PhD thesis 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, much like cutting them out with scissors. By applying this method, the Jena researchers succeeded in turning off the genes responsible for the fish’s pigmentation, thereby 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 picture of internal organs and their development inside a living animal.
Clearline currently includes approximately 200 animals at FLI, both male and female, which can now be used in numerous aging research programs.
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,” says Professor Englert. Senescent cells are cells that no longer divide and that affect surrounding cells and tissues by continuously releasing pro-inflammatory factors. So far, little is known about the role and function of these cells.
It is known that in humans, but also in mice or fish, the number of senescent cells increases with age, causing a kind of permanent inflammation in the body during the aging process. Targeted removal of senescent cells could 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 interest not only to the pharmaceutical and antiaging industry, but also to aging research.
“With the klara line, we can now investigate the role of senescent cells in the living organism at a molecular level. By labeling them with fluorophores and then examining them under a fluorescence microscope, we can learn where they occur in the body and whether they are likely to cluster in specific locations and what effects their removal has on the surrounding cells and tissues,” says Dr. Krug, highlighting the advantages of klara. This will lead to new insights into the role and function of this particular cell population during aging.
Johannes Krug et al, Generation of a transparent killifish line via CRISPR/Cas9-mediated multiple gene inactivation, eLife (2023). DOI: 10.7554/eLife.81549
Provided by the Fritz Lipmann Institute
Reference: 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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