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Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis.

Seruggia D, Fernández A, Cantero M, Pelczar P, Montoliu L - Nucleic Acids Res. (2015)

Bottom Line: Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines.The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus.Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Campus Cantoblanco, Darwin 3, 28049 Madrid, Spain CIBERER-ISCIII, Madrid, Spain.

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Inactivation of the Tyr 5′ boundary element at the endogenous locus and in modified YAC Tyr transgenes. (A) Distinct Tyr mouse models, both transgenic (YAC YRT4 (7); YAC ΔLCR (18) and genome-edited (TYRINS5) individuals, carrying the inactivation of the Tyr 5′ boundary element presented along with albino outbred HsdWin:NMRI mice and fully pigmented YAC YRT2 transgenic mice carrying the entire Tyr expression domain (63). The coat color of ΔLCR and YRT4 YAC transgenic mice is almost indistinguishable from albino mice due to a strongly variegated Tyr expression, but eyes are pigmented. In contrast, the coat color of the TYRINS5 edited mouse is light gray. (B) Whole-mounted retinae obtained from a TYRINS5 edited mouse. (C) Whole-mounted retinae obtained from a ΔLCR transgenic mouse. Variegation is observed in ΔLCR mice, at the RPE cell layer, whereas variegation is absent in TYRINS5 mice. In addition, a more severe loss of pigmentation at underlying choroidal melanocytes is observed in ΔLCR as compared with TYRINS5 mice. Scale bars in (B) and (C) panels correspond to 50 μm.
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Figure 5: Inactivation of the Tyr 5′ boundary element at the endogenous locus and in modified YAC Tyr transgenes. (A) Distinct Tyr mouse models, both transgenic (YAC YRT4 (7); YAC ΔLCR (18) and genome-edited (TYRINS5) individuals, carrying the inactivation of the Tyr 5′ boundary element presented along with albino outbred HsdWin:NMRI mice and fully pigmented YAC YRT2 transgenic mice carrying the entire Tyr expression domain (63). The coat color of ΔLCR and YRT4 YAC transgenic mice is almost indistinguishable from albino mice due to a strongly variegated Tyr expression, but eyes are pigmented. In contrast, the coat color of the TYRINS5 edited mouse is light gray. (B) Whole-mounted retinae obtained from a TYRINS5 edited mouse. (C) Whole-mounted retinae obtained from a ΔLCR transgenic mouse. Variegation is observed in ΔLCR mice, at the RPE cell layer, whereas variegation is absent in TYRINS5 mice. In addition, a more severe loss of pigmentation at underlying choroidal melanocytes is observed in ΔLCR as compared with TYRINS5 mice. Scale bars in (B) and (C) panels correspond to 50 μm.

Mentions: The comparison of genome-edited mouse lines carrying a targeted inactivation of the 5′ Tyr boundary (this work) with other mouse transgenic lines bearing similar deletions within large chromosome-type genomic transgenes reveals interesting but fundamental differences. The absence of the Tyr 5′ boundary element in YRT4 (7), or its targeted deletion in YRT5 (7) and ΔLCR (18) YAC transgenic mice, results in a strong reduction of Tyr gene expression in neural crest-derived melanocytes, as demonstrated by a dramatic reduction in coat color pigmentation (almost albino-like), associated with strongly variegated expression (9) (Figure 5A). In contrast, the reduced coat color phenotype observed in all targeted TYRINS5 lines is milder, uniformly light grey, and without signs of variegation (Figures 4A and 5A). Additionally, a more severe phenotype is observed in the eyes of YRT4 and ΔLCR YAC transgenic mice, where a profound loss of pigment is obvious in choroidal melanocytes and strong variegation is observed at the RPE cell layer (9,18) (Figure 5C). In contrast, the expression of Tyr at the RPE cell layer appears unaffected in TYRINS5 mice in all analyzed lines, without signs of variegation, and the loss of Tyr expression in the choroid is less marked (Figure 5B), as it can also be inferred from eye melanin content measurements (Figure 4D).


Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis.

Seruggia D, Fernández A, Cantero M, Pelczar P, Montoliu L - Nucleic Acids Res. (2015)

Inactivation of the Tyr 5′ boundary element at the endogenous locus and in modified YAC Tyr transgenes. (A) Distinct Tyr mouse models, both transgenic (YAC YRT4 (7); YAC ΔLCR (18) and genome-edited (TYRINS5) individuals, carrying the inactivation of the Tyr 5′ boundary element presented along with albino outbred HsdWin:NMRI mice and fully pigmented YAC YRT2 transgenic mice carrying the entire Tyr expression domain (63). The coat color of ΔLCR and YRT4 YAC transgenic mice is almost indistinguishable from albino mice due to a strongly variegated Tyr expression, but eyes are pigmented. In contrast, the coat color of the TYRINS5 edited mouse is light gray. (B) Whole-mounted retinae obtained from a TYRINS5 edited mouse. (C) Whole-mounted retinae obtained from a ΔLCR transgenic mouse. Variegation is observed in ΔLCR mice, at the RPE cell layer, whereas variegation is absent in TYRINS5 mice. In addition, a more severe loss of pigmentation at underlying choroidal melanocytes is observed in ΔLCR as compared with TYRINS5 mice. Scale bars in (B) and (C) panels correspond to 50 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Inactivation of the Tyr 5′ boundary element at the endogenous locus and in modified YAC Tyr transgenes. (A) Distinct Tyr mouse models, both transgenic (YAC YRT4 (7); YAC ΔLCR (18) and genome-edited (TYRINS5) individuals, carrying the inactivation of the Tyr 5′ boundary element presented along with albino outbred HsdWin:NMRI mice and fully pigmented YAC YRT2 transgenic mice carrying the entire Tyr expression domain (63). The coat color of ΔLCR and YRT4 YAC transgenic mice is almost indistinguishable from albino mice due to a strongly variegated Tyr expression, but eyes are pigmented. In contrast, the coat color of the TYRINS5 edited mouse is light gray. (B) Whole-mounted retinae obtained from a TYRINS5 edited mouse. (C) Whole-mounted retinae obtained from a ΔLCR transgenic mouse. Variegation is observed in ΔLCR mice, at the RPE cell layer, whereas variegation is absent in TYRINS5 mice. In addition, a more severe loss of pigmentation at underlying choroidal melanocytes is observed in ΔLCR as compared with TYRINS5 mice. Scale bars in (B) and (C) panels correspond to 50 μm.
Mentions: The comparison of genome-edited mouse lines carrying a targeted inactivation of the 5′ Tyr boundary (this work) with other mouse transgenic lines bearing similar deletions within large chromosome-type genomic transgenes reveals interesting but fundamental differences. The absence of the Tyr 5′ boundary element in YRT4 (7), or its targeted deletion in YRT5 (7) and ΔLCR (18) YAC transgenic mice, results in a strong reduction of Tyr gene expression in neural crest-derived melanocytes, as demonstrated by a dramatic reduction in coat color pigmentation (almost albino-like), associated with strongly variegated expression (9) (Figure 5A). In contrast, the reduced coat color phenotype observed in all targeted TYRINS5 lines is milder, uniformly light grey, and without signs of variegation (Figures 4A and 5A). Additionally, a more severe phenotype is observed in the eyes of YRT4 and ΔLCR YAC transgenic mice, where a profound loss of pigment is obvious in choroidal melanocytes and strong variegation is observed at the RPE cell layer (9,18) (Figure 5C). In contrast, the expression of Tyr at the RPE cell layer appears unaffected in TYRINS5 mice in all analyzed lines, without signs of variegation, and the loss of Tyr expression in the choroid is less marked (Figure 5B), as it can also be inferred from eye melanin content measurements (Figure 4D).

Bottom Line: Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines.The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus.Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Campus Cantoblanco, Darwin 3, 28049 Madrid, Spain CIBERER-ISCIII, Madrid, Spain.

Show MeSH
Related in: MedlinePlus