| dc.contributor.advisor | Pšenička, Martin | |
| dc.contributor.author | Vu Thi, Trang | |
| dc.date.accessioned | 2021-12-09T11:49:33Z | |
| dc.date.available | 2021-12-09T11:49:33Z | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-05-24 | |
| dc.identifier.uri | https://dspace.jcu.cz/handle/123456789/37446 | |
| dc.format | 47p | |
| dc.format | 47p | |
| dc.language.iso | eng | |
| dc.publisher | Jihočeská univerzita | cze |
| dc.rights | Bez omezení | |
| dc.subject | CRISPR/Cas9 | eng |
| dc.subject | primordial germ cells | eng |
| dc.subject | dead end | eng |
| dc.subject | knockout | eng |
| dc.subject | sterilization | eng |
| dc.subject | sturgeons | eng |
| dc.subject | sterlet. | eng |
| dc.title | Utilization of genome editing technology to knock out <i>dnd1</i> gene in sturgeons | cze |
| dc.title.alternative | "Utilization of genome editing technology to knock out
dnd1 gene in sturgeons" | eng |
| dc.type | diplomová práce | cze |
| dc.identifier.stag | 47916 | |
| dc.description.abstract-translated | 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. | eng |
| dc.date.accepted | 2017-06-13 | |
| dc.description.department | Fakulta rybářství a ochrany vod | cze |
| dc.thesis.degree-discipline | Fishery and Protection of Waters | cze |
| dc.thesis.degree-grantor | Jihočeská univerzita. Fakulta rybářství a ochrany vod | cze |
| dc.thesis.degree-name | Ing. | |
| dc.thesis.degree-program | Agricultural Specialization | cze |
| dc.description.grade | Dokončená práce s úspěšnou obhajobou | cze |
| dc.description.defence | Student Trang Vu Thi, MSc. presented her Master's thesis to the committee.
Assoc. Prof. Martin Kocour, the committee secretary, acquainted the committee both with the supervisor's and the opponent's reviews. The student then answered additional questions.
The topic of the thesis was discussed further. The student adequately and correctly answered questions asked by the members of the committee. The committee's assessment agreed with the suggestions of the evaluators (supervisor and opponent) and the evaluation of the defense is to be found bellow. | cze |
