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Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation

View Article: PubMed Central - PubMed

ABSTRACT

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.

No MeSH data available.


Genotype analysis of cyp19a1a mutants.(A) Mosaic F0 embryos showing shifted HRMA curves (green) with the uninjected control ones (red). (B) HRMA curves of the heterozygous F1 as compared with that of the WT individuals (red). (C and D) The representatives of TALEN and CRISPR-induced germline mutations. Sequences in shadow indicate the TALEN binding sites or CRISPR target sites. The sequences highlighted in blue are PAM sites for CRISPR targeting and those in red are inserted sequences. The gel images on the top are HMA assays on the representative mutants. Each lane is corresponding to a mutant sequence listed below the gel image. (E) Schematic illustration of the primers used for PCR demonstration of mutation. The primer P3 is specific to the mutated site. (F) PCR verification of mutant DNA with primers P3 and P5. Ef1a is the housekeeping gene as the control. The full-length gels are included in supplementary information.
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f1: Genotype analysis of cyp19a1a mutants.(A) Mosaic F0 embryos showing shifted HRMA curves (green) with the uninjected control ones (red). (B) HRMA curves of the heterozygous F1 as compared with that of the WT individuals (red). (C and D) The representatives of TALEN and CRISPR-induced germline mutations. Sequences in shadow indicate the TALEN binding sites or CRISPR target sites. The sequences highlighted in blue are PAM sites for CRISPR targeting and those in red are inserted sequences. The gel images on the top are HMA assays on the representative mutants. Each lane is corresponding to a mutant sequence listed below the gel image. (E) Schematic illustration of the primers used for PCR demonstration of mutation. The primer P3 is specific to the mutated site. (F) PCR verification of mutant DNA with primers P3 and P5. Ef1a is the housekeeping gene as the control. The full-length gels are included in supplementary information.

Mentions: Both TALEN and CRISPR/Cas9 approaches were able to generate indel mutations in cyp19a1a gene (HRMA, Fig. 1A and B; HMA, Fig. 1C and D), and we did not observe significant difference between the two approaches in terms of efficiency (data not shown). The loss of cyp19a1a gene was also confirmed by semi-quantitative PCR detection by using a mutant-specific primer (P3) (Fig. 1E and F).


Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation
Genotype analysis of cyp19a1a mutants.(A) Mosaic F0 embryos showing shifted HRMA curves (green) with the uninjected control ones (red). (B) HRMA curves of the heterozygous F1 as compared with that of the WT individuals (red). (C and D) The representatives of TALEN and CRISPR-induced germline mutations. Sequences in shadow indicate the TALEN binding sites or CRISPR target sites. The sequences highlighted in blue are PAM sites for CRISPR targeting and those in red are inserted sequences. The gel images on the top are HMA assays on the representative mutants. Each lane is corresponding to a mutant sequence listed below the gel image. (E) Schematic illustration of the primers used for PCR demonstration of mutation. The primer P3 is specific to the mutated site. (F) PCR verification of mutant DNA with primers P3 and P5. Ef1a is the housekeeping gene as the control. The full-length gels are included in supplementary information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120357&req=5

f1: Genotype analysis of cyp19a1a mutants.(A) Mosaic F0 embryos showing shifted HRMA curves (green) with the uninjected control ones (red). (B) HRMA curves of the heterozygous F1 as compared with that of the WT individuals (red). (C and D) The representatives of TALEN and CRISPR-induced germline mutations. Sequences in shadow indicate the TALEN binding sites or CRISPR target sites. The sequences highlighted in blue are PAM sites for CRISPR targeting and those in red are inserted sequences. The gel images on the top are HMA assays on the representative mutants. Each lane is corresponding to a mutant sequence listed below the gel image. (E) Schematic illustration of the primers used for PCR demonstration of mutation. The primer P3 is specific to the mutated site. (F) PCR verification of mutant DNA with primers P3 and P5. Ef1a is the housekeeping gene as the control. The full-length gels are included in supplementary information.
Mentions: Both TALEN and CRISPR/Cas9 approaches were able to generate indel mutations in cyp19a1a gene (HRMA, Fig. 1A and B; HMA, Fig. 1C and D), and we did not observe significant difference between the two approaches in terms of efficiency (data not shown). The loss of cyp19a1a gene was also confirmed by semi-quantitative PCR detection by using a mutant-specific primer (P3) (Fig. 1E and F).

View Article: PubMed Central - PubMed

ABSTRACT

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.

No MeSH data available.