Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: TALEN-induced AHR mutations. A, XLK-WG cells were transfected with TALEN pairs targeting helix 1 of the bHLH motif within exon 2 of AHR1α or AHR1β. Conserved functional or sequence domains are indicated by the following abbreviations: LBD, ligand-binding domain; TAD, transcriptional activation domain; PAS, Per-ARNT-Sim domain (Ma, 2012). TALEN binding signatures are underlined. *denotes sequence identities. B, Mutant AHRs were amplified with primers flanking the TALEN target site, and PCR products were cloned and sequenced. Genomic DNA sequences are depicted with TALEN-binding signatures highlighted. The predicted protein sequence of each mutant AHR was determined by translation of open reading frame. *indicates stop codon.

Article Snippet: TALEN transfection TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Transfection, Functional Assay, Sequencing, Ligand Binding Assay, Activation Assay, Binding Assay, Mutagenesis, Amplification, Clone Assay

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: Mutant AHRs are transcriptionally inactive in response to TCDD. COS-7 cells were treated with 100 nM TCDD or DMSO (0.6%) for 18 h beginning 5 h after transfection with AHR and ARNT constructs. Transcriptional activity was measured in triplicate using a luciferase reporter gene driven by a portion of the mouse CYP1A1 enhancer. Transactivation is given by the ratio of reporter-derived luciferase luminescence to that of constitutively expressed Renilla luciferase, reported as RLU. A, Detection of heterologously expressed AHRs by western blotting using antibody SA-210. B, Reporter gene activity. Cells were transfected with 50 ng ARNT and an equal amount of AHR or vector. n = 2 biological replicates. Representative experiment. Error bars = SEM. C, Co-expression of AHR1α (solid arrow) and AHR1β-B1 (open arrow) detected by western blotting. Labels above indicate quantities of each expression plasmid transfected per well. D, Reporter gene activity. Cells were transfected with 50 ng AHR1α expression plasmid and indicated amounts of AHR1β-B1 plasmid. n = 2 biological replicates. Representative experiment is presented. Error bars = SEM.

Article Snippet: TALEN transfection TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Mutagenesis, Transfection, Construct, Activity Assay, Luciferase, Derivative Assay, Western Blot, Plasmid Preparation, Expressing

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: AHR1α and AHR1β co-regulate CYP1A6 induction by TCDD. A, Cells were treated with 100 nM TCDD or DMSO (0.25%) for 22 h. RNA was harvested, reverse transcribed, and amplified in triplicate using qPCR with β-actin as endogenous control. Relative expression is normalized to mRNA abundance in DMSO-treated XLK-WG cells. Two-way ANOVA with Tukey’s multiple comparisons test. P = .03. n = 3 biological replicates. B, XLK-WG cells were treated with NTC siRNA or siAHR1β. Relative AHR1β expression was measured as described 30 h post-transfection. C, Cells were transfected with NTC or siAHR1β (300 nM) and treated with 100 nM TCDD or DMSO (0.5%) 8 h after transfection. RNA was harvested 30 h following transfection, and CYP1A6 was amplified as described. Two-way ANOVA with Tukey’s multiple comparisons test. P < .0001, n = 3 biological replicates. Error bars = SEM. *P < .01.

Article Snippet: TALEN transfection TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Reverse Transcription, Amplification, Control, Expressing, Transfection

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: FICZ induces AHR1α-mediated expression of a CYP1A1-driven reporter gene. COS-7 cells were transfected with 50 ng ARNT and 50 ng AHR or vector. Five hours after transfection, cells were treated with 100 nM FICZ or DMSO (0.6%) for 18 h. Transcriptional activity was measured in triplicate using a luciferase reporter gene driven by a portion of the mouse CYP1A1 enhancer. Transactivation is given by the ratio of reporter-derived luciferase luminescence to constitutively expressed control Renilla luciferase luminescence, reported as RLUs. n = 2 biological replicates. Representative experiment is depicted. Error bars = SEM.

Article Snippet: TALEN transfection TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Expressing, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Derivative Assay, Control

Journal: PLoS ONE

Article Title: Efficient TALEN Construction for Bombyx mori Gene Targeting

doi: 10.1371/journal.pone.0073458

Figure Lengend Snippet: Plasmid pCS2-TAL, pBlue-TAL and pBLTC contain a central region of 34 a.a. repeats (vertical hatch) flanked by N- terminal and C-terminal TAL sequences of different lengths (solid rectangles). SP6 and T7 indicate the SP6 and T7 promoters (open arrowheads), respectively. Arrows represent the Fok I nuclease domain, gray rectangles the 3′ and 5′ UTRs and rectangles with angled hatch the sequences encoding nuclear localization signals.

Article Snippet: TALEN constructs for the production of pBLTC TALEN was obtained from Cellectis Bioresearch (Paris, France).

Techniques: Plasmid Preparation

Journal: PLoS ONE

Article Title: Efficient TALEN Construction for Bombyx mori Gene Targeting

doi: 10.1371/journal.pone.0073458

Figure Lengend Snippet: (A) Positions of TALEN targets in the BmBLOS2 gene. Open rectangles and numbers above represent exons and their length in bp. The filled box is the 5′- UTR. The sequence above the line indicates part of exon 2 with BLTC target sites and their names. The sequence below the line indicates the third exon with BLT-2 and BLTS targets. Bgl II restriction site is underlined. The c above the Bgl II site indicates polymorphism in w1-pnd strain. (B) Positions of TALEN targets in the Bm-re gene. Open rectangles and numbers above represent exons and their lengths in bp. Filled boxes are 5′-and 3′ UTRs. The sequence below the line indicates the ninth exon (capital letters). Bgl II restriction site is underlined.

Article Snippet: TALEN constructs for the production of pBLTC TALEN was obtained from Cellectis Bioresearch (Paris, France).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Efficient TALEN Construction for Bombyx mori Gene Targeting

doi: 10.1371/journal.pone.0073458

Figure Lengend Snippet: The number of BLTS and BLTC mutants is underestimated due to a lower number of males detectable by phenotypic screening. Solid circles represent BLTS, BLT-2 and BLTC, whereas the open circle denotes the Bm-re TALEN.

Article Snippet: TALEN constructs for the production of pBLTC TALEN was obtained from Cellectis Bioresearch (Paris, France).

Techniques:

Journal: PLoS ONE

Article Title: Efficient TALEN Construction for Bombyx mori Gene Targeting

doi: 10.1371/journal.pone.0073458

Figure Lengend Snippet: (A) Mutant alleles obtained from the somatic assay; (B) Mutant alleles obtained from G 1 individuals. Sequences of mutant alleles are aligned with the respective wild-type sequences. TALEN target sites in the wild-type sequences are highlighted. The number of individuals carrying identical mutation is indicated to the right of each sequence.

Article Snippet: TALEN constructs for the production of pBLTC TALEN was obtained from Cellectis Bioresearch (Paris, France).

Techniques: Mutagenesis, Sequencing

Journal: PLoS ONE

Article Title: Efficient TALEN Construction for Bombyx mori Gene Targeting

doi: 10.1371/journal.pone.0073458

Figure Lengend Snippet: Embryos were photographed five days after Bm-re TALEN microinjection. (A) not injected control, (B) Bm-re mutant, (C, D) embryos injected with buffer, (E-H) G 0 embryos injected with TALEN mRNA.

Article Snippet: TALEN constructs for the production of pBLTC TALEN was obtained from Cellectis Bioresearch (Paris, France).

Techniques: Microinjection, Injection, Control, Mutagenesis

Journal: PLoS ONE

Article Title: Efficient TALEN Construction for Bombyx mori Gene Targeting

doi: 10.1371/journal.pone.0073458

Figure Lengend Snippet: The number of individuals carrying identical mutations is indicated to the right of each sequence. TALEN target sites in the wild-type sequences are boxed. (A) Mutant alleles from the somatic assay; (B) G 1 germline mutations.

Article Snippet: TALEN constructs for the production of pBLTC TALEN was obtained from Cellectis Bioresearch (Paris, France).

Techniques: Sequencing, Mutagenesis

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: PCR Primers

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques:

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: TALEN-induced AHR mutations. A, XLK-WG cells were transfected with TALEN pairs targeting helix 1 of the bHLH motif within exon 2 of AHR1α or AHR1β. Conserved functional or sequence domains are indicated by the following abbreviations: LBD, ligand-binding domain; TAD, transcriptional activation domain; PAS, Per-ARNT-Sim domain (Ma, 2012). TALEN binding signatures are underlined. *denotes sequence identities. B, Mutant AHRs were amplified with primers flanking the TALEN target site, and PCR products were cloned and sequenced. Genomic DNA sequences are depicted with TALEN-binding signatures highlighted. The predicted protein sequence of each mutant AHR was determined by translation of open reading frame. *indicates stop codon.

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Transfection, Functional Assay, Sequencing, Ligand Binding Assay, Activation Assay, Binding Assay, Mutagenesis, Amplification, Clone Assay

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: AHR expression in mutant strains. A, qPCR. Total RNA was harvested from wild-type cells and reverse transcribed. mRNA abundance was measured in triplicate using qPCR with β-actin as endogenous control. n = 3. Representative experiment is depicted. B, Western blot. Cell lysate was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with primary antibody SA-210 (1:1000; Enzo) directed against the N-terminal region of AHR1α (94.2 kDa) and AHR1β (93.6 kDa). AHR protein standards were generated by in vitro transcription using the TNT Quick-coupled Transcription/Translation kit (Promega). C and D, qPCR was accomplished as described. mRNA expression is reported relative to wild-type cells. C, AHR1α. One-way ANOVA with Tukey’s multiple comparisons test. p = .043. n = 5 biological replicates. D, AHR1β. One-way ANOVA with Tukey’s multiple comparisons test. P = .011. n = 4 biological replicates. Error bars = SEM. *P < .05.

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Expressing, Mutagenesis, Western Blot, SDS Page, Generated, In Vitro

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: Mutant AHRs are transcriptionally inactive in response to TCDD. COS-7 cells were treated with 100 nM TCDD or DMSO (0.6%) for 18 h beginning 5 h after transfection with AHR and ARNT constructs. Transcriptional activity was measured in triplicate using a luciferase reporter gene driven by a portion of the mouse CYP1A1 enhancer. Transactivation is given by the ratio of reporter-derived luciferase luminescence to that of constitutively expressed Renilla luciferase, reported as RLU. A, Detection of heterologously expressed AHRs by western blotting using antibody SA-210. B, Reporter gene activity. Cells were transfected with 50 ng ARNT and an equal amount of AHR or vector. n = 2 biological replicates. Representative experiment. Error bars = SEM. C, Co-expression of AHR1α (solid arrow) and AHR1β-B1 (open arrow) detected by western blotting. Labels above indicate quantities of each expression plasmid transfected per well. D, Reporter gene activity. Cells were transfected with 50 ng AHR1α expression plasmid and indicated amounts of AHR1β-B1 plasmid. n = 2 biological replicates. Representative experiment is presented. Error bars = SEM.

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Mutagenesis, Transfection, Construct, Activity Assay, Luciferase, Derivative Assay, Western Blot, Plasmid Preparation, Expressing

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: AHR1α and AHR1β co-regulate CYP1A6 induction by TCDD. A, Cells were treated with 100 nM TCDD or DMSO (0.25%) for 22 h. RNA was harvested, reverse transcribed, and amplified in triplicate using qPCR with β-actin as endogenous control. Relative expression is normalized to mRNA abundance in DMSO-treated XLK-WG cells. Two-way ANOVA with Tukey’s multiple comparisons test. P = .03. n = 3 biological replicates. B, XLK-WG cells were treated with NTC siRNA or siAHR1β. Relative AHR1β expression was measured as described 30 h post-transfection. C, Cells were transfected with NTC or siAHR1β (300 nM) and treated with 100 nM TCDD or DMSO (0.5%) 8 h after transfection. RNA was harvested 30 h following transfection, and CYP1A6 was amplified as described. Two-way ANOVA with Tukey’s multiple comparisons test. P < .0001, n = 3 biological replicates. Error bars = SEM. *P < .01.

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Amplification, Expressing, Transfection

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: AHR1α and AHR1β differentially regulate transcription in response to TCDD. Cells were treated with 100 nM TCDD or DMSO (0.25%). Twenty-two hours after exposure, RNA was harvested, reverse transcribed, and amplified in triplicate using qPCR with β-actin as endogenous control. Relative expression is normalized to mRNA abundance in DMSO-treated XLK-WG cells. A, Two-way ANOVA with Tukey’s multiple comparisons test. P = .043. n = 3 biological replicates. B, Two-way ANOVA with Tukey’s multiple comparisons test. P = .007, n = 3 biological replicates. C, Two-way ANOVA with Tukey’s multiple comparisons test. P = .02. n = 4 biological replicates. Error bars = SEM. *P < .05.

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Amplification, Expressing

Journal: Toxicological Sciences

Article Title: Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis : Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

doi: 10.1093/toxsci/kfw212

Figure Lengend Snippet: AHR1α and AHR1β have distinct target gene preferences during FICZ responsiveness. Cells were treated with 100 nM FICZ or DMSO (0.25%). Twenty-two hours after exposure, RNA was harvested, reverse transcribed, and amplified in triplicate using qPCR with β-actin as endogenous control. Relative expression is normalized to mRNA abundance in DMSO-treated XLK-WG cells. A, Two-way ANOVA with Tukey’s multiple comparisons test. P = .009. n = 3 biological replicates. B, Two-way ANOVA with Tukey’s multiple comparisons test. P = .001, n = 4 biological replicates. Error bars = SEM. *P < .05.

Article Snippet: TALEN constructs engineered to target exon 2 of AHR1 α or AHR1β were obtained from Cellectis BioResearch (Paris, France), each in the pTAL.CMV.T7.v2 vector (Details in Supplementary Materials ).

Techniques: Amplification, Expressing