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.
Klíčová slova
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Klíčová slova v angličtině
CRISPR/Cas9, primordial germ cells, dead end, knockout, sterilization, sturgeons, sterlet.
Rozsah průvodní práce
47p
Jazyk
AN
Anotace
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Anotace v angličtině
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.
Klíčová slova
-
Klíčová slova v angličtině
CRISPR/Cas9, primordial germ cells, dead end, knockout, sterilization, sturgeons, sterlet.
Zásady pro vypracování
Primordial germ cells (PGCs) are embryonic precursors of gametes. They migrate from the place of their origin into genital ridge, where they develop into gonial cells, undergo proliferation, differentiation and recombination of genetic information during gametogenesis. The function of dead end (dnd1) gene has been described well in model species, and in zebrafish it is crucial for the migration and development of PGCs. At early stages of embryonic development, dnd1 is maternally supplied and later the dnd1 transcript is produced by germ stem cells until differentiation. However, little is known about its function in several other species. Sturgeon, as representative of fish with holoblastic cleavage pattern of embryo, has a completely different origin of PGCs as well as deposits of maternally supplied dnd1 in comparison with teleostean fishes. Therefore, it will be interesting to reveal more about the function/s of dnd1 in these ancient species. Recently, by using antisense morpholino oligonucleotides we have found that the inhibition of translation of maternally supplied RNA led to un-directional migration and depletion of PGCs in sturgeons. In the present study, we aim to extend the state-of-art and target directly DNA in order to elucidate the function of dnd1 transcribed from zygotic genome after migration of PGCs. For this purpose, we will use CRISPR/CAS9, a cutting-edge genome editing technology that presents certain advantages over other genome editing technologies such as TALENS and ZFNs. This technology has been successfully used in various animal models, and in zebrafish, the CRISPR/CAS9 can generate gene knockout with very high efficiency. It is assumed that knock out of dnd1 will lead to sterilization of treated animals; hence they can be practically used as recipients for technology of surrogate production. In addition, this will be a pioneer study on utilization of recent techniques of genome editing technology, CRISPR/Cas9, in sturgeons.
Zásady pro vypracování
Primordial germ cells (PGCs) are embryonic precursors of gametes. They migrate from the place of their origin into genital ridge, where they develop into gonial cells, undergo proliferation, differentiation and recombination of genetic information during gametogenesis. The function of dead end (dnd1) gene has been described well in model species, and in zebrafish it is crucial for the migration and development of PGCs. At early stages of embryonic development, dnd1 is maternally supplied and later the dnd1 transcript is produced by germ stem cells until differentiation. However, little is known about its function in several other species. Sturgeon, as representative of fish with holoblastic cleavage pattern of embryo, has a completely different origin of PGCs as well as deposits of maternally supplied dnd1 in comparison with teleostean fishes. Therefore, it will be interesting to reveal more about the function/s of dnd1 in these ancient species. Recently, by using antisense morpholino oligonucleotides we have found that the inhibition of translation of maternally supplied RNA led to un-directional migration and depletion of PGCs in sturgeons. In the present study, we aim to extend the state-of-art and target directly DNA in order to elucidate the function of dnd1 transcribed from zygotic genome after migration of PGCs. For this purpose, we will use CRISPR/CAS9, a cutting-edge genome editing technology that presents certain advantages over other genome editing technologies such as TALENS and ZFNs. This technology has been successfully used in various animal models, and in zebrafish, the CRISPR/CAS9 can generate gene knockout with very high efficiency. It is assumed that knock out of dnd1 will lead to sterilization of treated animals; hence they can be practically used as recipients for technology of surrogate production. In addition, this will be a pioneer study on utilization of recent techniques of genome editing technology, CRISPR/Cas9, in sturgeons.
Seznam doporučené literatury
Linhartová, Z., Saito, T., Kašpar, V., Rodina, M., Práková, E., Hagihara, S., Pšenička, M., 2015. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene. Theriogenology 84, 1246-1255. doi:10.1016/j.theriogenology.2015.07.003
Saito, T., Pšenička, M., 2015. Novel Technique for Visualizing Primordial Germ Cells in Sturgeons (Acipenser ruthenus, A. gueldenstaedtii, A. baerii, and Huso huso). Biol. Reprod. 93, 96. doi:10.1095/biolreprod.115.128314
Saito, T., Pšenička, M., Goto, R., Adachi, S., Inoue, K., Arai, K., Yamaha, E., 2014. The origin and migration of primordial germ cells in sturgeons. PLoS One 9. doi:10.1371/journal.pone.0086861
Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B., Raz, E., 2003. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr. Biol. 13, 1429-1434. doi:10.1016/S0960-9822(03)00537-2
Yang, X., Yue, H., Ye, H., Li, C., Wei, Q., 2015. Identification of a germ cell marker gene, the dead end homologue, in Chinese sturgeon Acipenser sinensis. Gene 558, 118-25. doi:10.1016/j.gene.2014.12.059
Hwang, WY., Fu, Y., Reyon, D., Maeder, ML., Tsai, SQ., Sander, JD., Peterson, RT., Yeh, JR., Joung, JK., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227-9. doi: 10.1038/nbt.2501.
Sander, JD., Joung, JK., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347-55. doi: 10.1038/nbt.2842.
Seznam doporučené literatury
Linhartová, Z., Saito, T., Kašpar, V., Rodina, M., Práková, E., Hagihara, S., Pšenička, M., 2015. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene. Theriogenology 84, 1246-1255. doi:10.1016/j.theriogenology.2015.07.003
Saito, T., Pšenička, M., 2015. Novel Technique for Visualizing Primordial Germ Cells in Sturgeons (Acipenser ruthenus, A. gueldenstaedtii, A. baerii, and Huso huso). Biol. Reprod. 93, 96. doi:10.1095/biolreprod.115.128314
Saito, T., Pšenička, M., Goto, R., Adachi, S., Inoue, K., Arai, K., Yamaha, E., 2014. The origin and migration of primordial germ cells in sturgeons. PLoS One 9. doi:10.1371/journal.pone.0086861
Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B., Raz, E., 2003. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr. Biol. 13, 1429-1434. doi:10.1016/S0960-9822(03)00537-2
Yang, X., Yue, H., Ye, H., Li, C., Wei, Q., 2015. Identification of a germ cell marker gene, the dead end homologue, in Chinese sturgeon Acipenser sinensis. Gene 558, 118-25. doi:10.1016/j.gene.2014.12.059
Hwang, WY., Fu, Y., Reyon, D., Maeder, ML., Tsai, SQ., Sander, JD., Peterson, RT., Yeh, JR., Joung, JK., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227-9. doi: 10.1038/nbt.2501.
Sander, JD., Joung, JK., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347-55. doi: 10.1038/nbt.2842.
Přílohy volně vložené
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Přílohy vázané v práci
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Převzato z knihovny
Ano
Plný text práce
Přílohy
Posudek(y) oponenta
Hodnocení vedoucího
Záznam průběhu obhajoby
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.