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Utilization of genome editing technology to knock out \kur{dnd1} gene in sturgeons
VU THI, Trang
In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.
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Molecular basis of the Amphibian germ plasm
Špirhanzlová, Petra ; Krylov, Vladimír (advisor) ; Nedvídek, Josef (referee)
2. Abstract Xenopus germ plasm originates in previtellogenic oocytes in an area rich in mitochondria - the mitochondrial cloud. After its first appearance, the germ plasm localises to the vegetative pole of the egg and takes the form of small islands. After fertilization, the germ plasm aggregates at the apex of the vegetal pole of the embryo and then it separates into blastomers directly surrounding the vegetal pole. The germ plasm consists of mitochondria, germinal granules, ribozomes and various ribonucleic acids (RNAs). In Xenopus, the germ plasm RNAs are required for the formation of the primordial germ cell line. XDead end is fundamental for the migration, differentiation and survival of germ cells. Embryos depleted of fatvg are defective in primordial germ cell (PGC) formation and cortical rotation and organelle transport are inhibited in these embryos. Xdazl is required for early PGC differentiation and indirectly contributes to the migration of PGCs through the endoderm. Some germ plasm proteins, for example, Fatvg and Xcat-2, also play an important role in the determination of the dorsal/ventral axis.
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