Zebrafish, Medaka And Turquoise Killifish For Understanding Human Neurodegenerative/Neurodevelopmental Disorders Part 3

3.2. Ease of Gene Editing with Small Fishes

It is estimated that approximately 71% of human genes have at least one zebrafish gene orthologue [31]. 

The relationship between human genes and memory has always been a topic of great concern. Scientific research shows that human genes have a very important impact on our memory. Genes are naturally inherited information in the human body and are passed on to future generations through inheritance. In this process, genes influence our physical characteristics and functions, including our memory.

First, scientists have confirmed the relationship between some genes and memory. For example, research on certain genes shows that they play a key role in influencing learning and memory in people's brains. These genes can influence communication between neurons in the brain as well as the structure and function of the brain.

In addition, research also shows that genes may also be related to the development of memory. This means that some people have better memories than others, and this may be due to the good genes they inherit.

Although genes play an important role in influencing memory, they are not the only factor. Our environment and experiences can also affect our memory. Our brains can improve memory through continuous learning and training, which is also very beneficial to our brain activity and health.

Therefore, we should maintain a positive attitude and engage in effective exercise and study to improve our memory. At the same time, we can also refer to our genes to understand our strengths and weaknesses to better plan our lives and work. The most important thing is that we should maintain an optimistic attitude and always believe that we can have better memory, which will help us better achieve our goals and desires.

In summary, the relationship between human genes and memory is very complex. While genes play an important role in influencing memory, our environment and experiences are equally important. Let us actively participate in exercise and learning, unleash our potential, and create our memory miracles. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the multiple active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

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In addition, approximately 84% of the genes known to be associated with human diseases have orthologues in the zebrafish genome [32]. 

Small fishes have the advantage of simple procedures for gene editing. Efficient genome engineering can be achieved by zinc-finger nucleases (ZFNs) or transcription activator-like effectors (TALENs) in various model organisms, including zebrafish and medaka [33–36], and the emergence of the CRISPR-Cas9 technique has made it much easier to knock out or edit specific genes in recent years [37–39]. 

In small fishes, oviposition and fertilization occur outside the parent's body, and gene editing factors are introduced into the fertilized eggs by microinjection, which can then hatch in a petri dish [40]. 

For example, by injecting GFP mRNA into the embryo, it is possible to observe the results the day after the injection without waiting for days or months [41]. Improved and advanced techniques for gene editing have been reported thus far, and the highly mutagenic CRISPR-Cas9 method enables even injection of F0 embryos mimicking null mutants, making the induction of mutations quick and efficient [42]. 

Morpholino antisense oligonucleotide-based knockdown of target genes has often been conducted in small fishes, but there can be phenotypic discrepancies between morpholino and knockout phenotypes [43]. This may be due to the presence of morpholino-induced off-target effects or the presence of nonsense-mediated mRNA decay and associated genetic compensation in knockout individuals [44]. 

In the analysis of gene function in zebrafish, it is strongly recommended to generate mutants and analyze their phenotypes instead of using antisense morpholino oligonucleotides. The CRISPR-Cas9 system also enables knock-in of the target gene or specific gene variants using homology-directed repair (HDR) or other mechanisms [45–50]. 

The stable introduction of exogenous DNA in the zebrafish genome was already achieved in 1988 [51]. Coinjection of the I-SceI meganuclease enzyme together with the transgenic vector carrying the restriction sites [52] or coinjection of tol2 or sleeping beauty transposase with the transgenic vector carrying the transgenic cassette flanked by cis-regulatory repeats [53,54] facilitates germline transmission of the transgene very effectively. 

These techniques of genome engineering apply to zebrafish, medaka, and turquoise killifish, but we think it is difficult to conduct microinjection into the eggs of turquoise killifish. This is because of the relatively hard chorion of turquoise killifish compared to zebrafish and medaka, and please kindly see the excellent publications about genome engineering of turquoise killifish [55,56].

If the same thing were to be done with the mouse, gene editing of the embryo would be a complex operation that would require the collection of fertilized eggs from the mated female mouse body, microinjection of the genome-engineering solution into the eggs under clean operations, and implantation of the injected eggs into the fallopian tubes of a pseudopregnant mouse, which would be technically much more difficult and expensive. 

Zebrafish can produce more than 100 eggs per oviposition, making it easy to pick up individuals with the desired gene editing. Zebrafish will mature into adults in 3 months, making it possible to breed the next generation. 

In the case of medaka and turquoise killifish, the number of eggs per oviposition seems smaller than that of zebrafish, but they lay eggs every day if the condition of the adult fish and aquarium is ideal. 

Medaka and turquoise killifish can show sexual maturation in approximately 2–3 months and 1 month, respectively. Compared to mice, the cost of subsequent management can also be greatly reduced. As described above, zebrafish and medaka are the mainstream models for genetic manipulation, while the turquoise killifish provides an excellent model for the studies of aging and age-related disorders (Figure 1). 

The aging of turquoise killifish is described in the next chapter. When designing a genetic study using small fishes, it is necessary to be conscious of several genetic features. 

Zebrafish are diploid with 25 chromosomes (25 × 2n) and are close to humans, but their sex chromosomes have not been identified. The mechanism of sex determination is still not fully uncovered, but it has been found that zebrafish sex can be affected by environmental factors [57–59]. In contrast, medaka or turquoise killifish sex is determined by XX/XY sex chromosomes [60–62]. 

Vertebrates, including humans, experienced a whole-genome duplication in which the genome doubled twice in our ancestors approximately 500 million years ago. Furthermore, teleosts, including zebrafish, medaka, and turquoise killifish, experienced another round of whole-genome duplication [63–65]. 

Therefore, in some genes, there is no 1:1 correspondence between humans and zebrafish, and there can be two orthologous genes. The fact that there are two orthologues means that even if one of the orthologues is knocked out, the other paralogue may be able to compensate for its function. 

It has also been reported that there are differences in the structure, function, and expression patterns between the paralogues of these genes, which might suggest that each gene has its own significance [66]. 

In some cases, a double knockout of those two orthologous genes might be necessary to establish a knockout line with the anticipated phenotypes seen in human diseases or disorders. It is also necessary to check whether the ortholog of the gene of interest exists or not (e.g., the ortholog of SNCA is present in medaka but not in zebrafish).

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