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Použití CRIPR/Cas9 a nové techniky značení zárodečných buněk pro náhradní reprodukci u jeseterů
KHANZAI BALOCH, Abdul Rasheed
Sturgeons are commonly known as living fossils or ancient giants that diverged from ancient pre-Jurassic teleost lineage approximately ~300 million years ago (Mya). Sturgeons' 85% species are listed as critically endangered in the International Union for Conservation of Nature (IUCN). Sturgeons' reproductive traits such as delay in sexual maturation and periodic interrupted spawning cycles make their rehabilitation more difficult. However, among sturgeon species, the sterlet (Acipenser ruthenus) has shortest sexual maturation period. Therefore, it can be used as a host in surrogate production in sturgeons. Dnd1 was discovered as germ-plasm specific maternal RNA that exclusively expresses in vertebrate germ-line. Various studies have confirmed that dnd1 protein is essential for Primordial Germ Cells (PGCs) migration; and disruption of the PGCs migration affects fish fertility. Dnd1 deficient PGCs in zebrafish transdifferentiate into somatic cells. Previously our colleagues used morpholino oligonucleotide to knock down dnd1 in sterlet to produce germ cell free host for surrogate production. CRISPR/Cas9, a cutting-edge genome editing technology is being used in different research fields; here we thus aimed to harness the power of aforementioned technology to knock out dnd1 in sterlet. No or less number of PGCs were observed in CRISPR/Cas9 injected embryos as compared to control group injected with FITC-dextran only in order to label PGCs. Furthermore, we compared three different sterilization techniques viz., CRISPR/Cas9 and morpholino oligonucleotide (MO) targeting dnd1 and ultraviolet irradiation to eliminate PGCs in sterlet. Our data showed higher hatching and survival rates in CRISPR/Cas9, UV irradiation, and MO knockdown groups, respectively. Interestingly, some embryos treated with CRISPR/Cas9 displayed malformations. We presume that malformations were due to off-target effects and/or due to double injections i.e., injection of CRISPR/Cas9 at animal pole to knock-out the dnd1 and FITC-dextran at vegetal pole. Taking advantages of Iron Oxide nanoparticles (IONs) applications in various burgeoning research fields, we opted to use them to label PGCs in sturgeons. We injected IONs combined with FITC-dextran into vegetal pole of sturgeon embryos, and have successfully labelled the PGCs. Injection of IONs in sturgeons did not affect hatching and survival rates of embryos. Interestingly at 5 dpf, significantly less number of FITC-dextran labelled PGCs in FITC-dextran/IONs injected group were observed when compared with PGCs that were labelled with FITC-dextran only. Less number of PGCs in IONs injected group presumably could be because of interference posed by IONs to PGCs during the course of their migration. This is first study of its kind where germ cells of any species have been labelled by using nanoparticles. In conclusions, this thesis provides information regarding role of Dnd1 protein as potential germ-cell molecular marker in various fish species, and its use for conservation of fish species. Dnd1 knockout sterlet can be potentially used as sterile host for surrogate production in sturgeons. Moreover, labelling of PGCs in sturgeons by using IONs can thus open new avenues to study interactions of nanoparticles with cells that will ultimately help in hyperthermia where cells/tissues are exposed to electromagnetic field increasing temperatures to activate their death. After insertion of IONs to PGC in sturgeon embryo, it could be possible to isolate PGC using a magnetic field or to apply hyperthermia for host sterilization purpose.
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Methods of rearing the early stages of sturgeon fishes
KAHANEC, Martin
The review "Methods of rearing the early stages of sturgeon fishes" brings in its introductory part an information on current systematics of chondrosteans. It has recently gone through certain amendments upon the development of genetic mapping the fish populations in open waters, as well as the specimens from fish farms. Owing to worldwide- and long-term reduction of stocks of the last populations of chondrostean fishes in open waters, controlled reproduction followed by progeny nursing is the only one real possibility to save chondrosteans. Reproduction of broodstock with incubation of fertilized eggs in fish hatcheries is relatively well managed but the transfer of swimming-up larvae to exogenic feeding is species-specific and very demanding in this group of fish, and frequently attended by remarkable losses of specimens, namely due to high mortality of early stages of fish. The thesis deals with basic currently performed methods of initial feeding of sturgeons. The respective types of live food and artificial feeds which are appropriate and available on the territory of the Czech Republic, are described in details. Furthermore, the thesis brings description of the basic technological parameters of fish farming facilities and technologies, as well as physical and chemical properties of water, which are the essential part of successful rearing the early stages of fish.
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