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Transnetyx genomic pcr
CNOT3 C-Terminal Domain Is Required for ESC Maintenance (A) Domain structure of mouse CNOT3. (B and C) Induction of Cnot3 deletion in Cnot3 cKO ESCs. Cells were treated with or without 4-OHT, and Cnot3 expression was determined by <t>qRT-PCR</t> (B) and western blot (C) at the indicated time points. Values were plotted as mean ± SEM from three independent experiments. (D) Interaction between CNOT3 fragments and CNOT1 or CNOT2. HA-tagged CNOT3 fragments were expressed in Cnot3 cKO ESCs and affinity purified by HA beads. Co-purified endogenous CNOT1 and CNOT2 were detected by western blot. Whole images of the same blots are shown in Figure S4 B. (E–G) Rescue of the deletion phenotype by the overexpression of CNOT3 domains. Cnot3 cKO ESCs expressing various CNOT3 fragments were treated with or without 4-OHT. Changes in cellular morphology (E; scale bars, 200 μm), marker expression (F), and colony formation (G) were determined by imaging, qRT-PCR, and alkaline phosphatase staining, respectively. For qRT-PCR, relative expression was normalized by Actin and plotted as mean ± SEM from three independent experiments.
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1) Product Images from "CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State"

Article Title: CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2016.09.007

CNOT3 C-Terminal Domain Is Required for ESC Maintenance (A) Domain structure of mouse CNOT3. (B and C) Induction of Cnot3 deletion in Cnot3 cKO ESCs. Cells were treated with or without 4-OHT, and Cnot3 expression was determined by qRT-PCR (B) and western blot (C) at the indicated time points. Values were plotted as mean ± SEM from three independent experiments. (D) Interaction between CNOT3 fragments and CNOT1 or CNOT2. HA-tagged CNOT3 fragments were expressed in Cnot3 cKO ESCs and affinity purified by HA beads. Co-purified endogenous CNOT1 and CNOT2 were detected by western blot. Whole images of the same blots are shown in Figure S4 B. (E–G) Rescue of the deletion phenotype by the overexpression of CNOT3 domains. Cnot3 cKO ESCs expressing various CNOT3 fragments were treated with or without 4-OHT. Changes in cellular morphology (E; scale bars, 200 μm), marker expression (F), and colony formation (G) were determined by imaging, qRT-PCR, and alkaline phosphatase staining, respectively. For qRT-PCR, relative expression was normalized by Actin and plotted as mean ± SEM from three independent experiments.
Figure Legend Snippet: CNOT3 C-Terminal Domain Is Required for ESC Maintenance (A) Domain structure of mouse CNOT3. (B and C) Induction of Cnot3 deletion in Cnot3 cKO ESCs. Cells were treated with or without 4-OHT, and Cnot3 expression was determined by qRT-PCR (B) and western blot (C) at the indicated time points. Values were plotted as mean ± SEM from three independent experiments. (D) Interaction between CNOT3 fragments and CNOT1 or CNOT2. HA-tagged CNOT3 fragments were expressed in Cnot3 cKO ESCs and affinity purified by HA beads. Co-purified endogenous CNOT1 and CNOT2 were detected by western blot. Whole images of the same blots are shown in Figure S4 B. (E–G) Rescue of the deletion phenotype by the overexpression of CNOT3 domains. Cnot3 cKO ESCs expressing various CNOT3 fragments were treated with or without 4-OHT. Changes in cellular morphology (E; scale bars, 200 μm), marker expression (F), and colony formation (G) were determined by imaging, qRT-PCR, and alkaline phosphatase staining, respectively. For qRT-PCR, relative expression was normalized by Actin and plotted as mean ± SEM from three independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Affinity Purification, Purification, Over Expression, Marker, Imaging, Staining

Cnot3 Is Required for Early Embryonic Development (A and B) Cnot3 expression in pre-implantation embryos. Expression was determined by qRT-PCR and plotted as mean ± SEM from three independent experiments (A) and immunofluorescence staining (B). Scale bar, 20 μm. (C) Immunofluorescence staining of CNOT3 in WT and Cnot3 deletion embryos at the indicated developmental stages. Scale bars, 20 μm. (D) Morphology of WT and Cnot3 deletion embryos at E6.5 and E7.5. Scale bars, 100 μm. (E) Morphology and OCT4 expression of Cnot3 deletion embryo at E6.5. Scale bars, 20 μm. (F) Numbers and genotypes of embryos collected at the indicated developmental stages. Numbers of morphologically abnormal embryos are listed in parentheses.
Figure Legend Snippet: Cnot3 Is Required for Early Embryonic Development (A and B) Cnot3 expression in pre-implantation embryos. Expression was determined by qRT-PCR and plotted as mean ± SEM from three independent experiments (A) and immunofluorescence staining (B). Scale bar, 20 μm. (C) Immunofluorescence staining of CNOT3 in WT and Cnot3 deletion embryos at the indicated developmental stages. Scale bars, 20 μm. (D) Morphology of WT and Cnot3 deletion embryos at E6.5 and E7.5. Scale bars, 100 μm. (E) Morphology and OCT4 expression of Cnot3 deletion embryo at E6.5. Scale bars, 20 μm. (F) Numbers and genotypes of embryos collected at the indicated developmental stages. Numbers of morphologically abnormal embryos are listed in parentheses.

Techniques Used: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Cnot3 Deletion Increases Differentiation Gene mRNA Poly(A) Tail Lengths (A) Schematic drawing for mRNA fractionation based on poly(A) tail length. (B) Validation of the oligo(dT) fractionation method. Cnot3 cKO ESCs were treated with (KO) or without 4-OHT (WT) for 48 hr. The poly(A) standards were mixed with total RNAs extracted from the cells, and RNAs were fractionated by oligo(dT) beads. The distribution of each standard in each fraction (A0, A1, A2) was determined by qRT-PCR and plotted as mean ± SEM from three independent experiments. (C) Measurements of poly(A) tail length for the indicated genes by oligo(dT) fractionation. (D) Examination of mRNA stability for the indicated genes. Cnot3 cKO ESCs were treated with (KO) or without 4-OHT (WT) for 48 hr. Actinomycin D was added to the cells, and mRNA level was measured by qRT-PCR at the indicated time points. Relative expression values were plotted as mean ± SEM from three independent experiments. (E) Proposed model. CNOT3-dependent differentiation gene mRNA deadenylation and degradation plays a critical role in the maintenance of the pluripotent state. ∗ p
Figure Legend Snippet: Cnot3 Deletion Increases Differentiation Gene mRNA Poly(A) Tail Lengths (A) Schematic drawing for mRNA fractionation based on poly(A) tail length. (B) Validation of the oligo(dT) fractionation method. Cnot3 cKO ESCs were treated with (KO) or without 4-OHT (WT) for 48 hr. The poly(A) standards were mixed with total RNAs extracted from the cells, and RNAs were fractionated by oligo(dT) beads. The distribution of each standard in each fraction (A0, A1, A2) was determined by qRT-PCR and plotted as mean ± SEM from three independent experiments. (C) Measurements of poly(A) tail length for the indicated genes by oligo(dT) fractionation. (D) Examination of mRNA stability for the indicated genes. Cnot3 cKO ESCs were treated with (KO) or without 4-OHT (WT) for 48 hr. Actinomycin D was added to the cells, and mRNA level was measured by qRT-PCR at the indicated time points. Relative expression values were plotted as mean ± SEM from three independent experiments. (E) Proposed model. CNOT3-dependent differentiation gene mRNA deadenylation and degradation plays a critical role in the maintenance of the pluripotent state. ∗ p

Techniques Used: Fractionation, Quantitative RT-PCR, Expressing

Related Articles

Polymerase Chain Reaction:

Article Title: CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State
Article Snippet: .. The genotypes of the animals were determined by Southern blot, genomic PCR (see for primer sequences), or qRT-PCR by Transnetyx. ..

Article Title: Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst
Article Snippet: .. The genotypes of mice used for breeding were determined by quantitative genomic PCR (Transnetyx) using primers specific to Hvcn1 intron 3 and pGT0Lxf ; results from the Transnetyx genotyping strategy were verified by triplex genomic PCR. .. Although quantitative RT-PCR confirmed that the RRN293 transgene was expressed at the expense of the wt Hvcn1 allele , we were unable to detect either β-galactosidase protein [using several different anti-β-galactosidase antibodies in Western blot analysis, immunocytochemistry (ICC) or immunohistochemistry (IHC)] or activity (using X-gal staining) in spleen or BMC from Hv1 −/− mice.

Article Title: Optogenetic Stimulation of Cholinergic Brainstem Neurons During Focal Limbic Seizures: Effects on Cortical Physiology
Article Snippet: .. Pups were genotyped using genomic PCR (Transnetyx). .. To generate cell-type specific expression of ChR2 in cholinergic neurons, we used a Cre-lox approach, by injecting a Cre-inducible recombinant AAV vector containing ChR2 (pAAV-Ef1α-DIO-hChR2(H134R)-EYFP-WPRE-pA) into rats expressing Cre-recombinase in cholineacetyltransferase (ChAT)-positive neurons ( ) .

Southern Blot:

Article Title: CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State
Article Snippet: .. The genotypes of the animals were determined by Southern blot, genomic PCR (see for primer sequences), or qRT-PCR by Transnetyx. ..

Quantitative RT-PCR:

Article Title: CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State
Article Snippet: .. The genotypes of the animals were determined by Southern blot, genomic PCR (see for primer sequences), or qRT-PCR by Transnetyx. ..

Mouse Assay:

Article Title: Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst
Article Snippet: .. The genotypes of mice used for breeding were determined by quantitative genomic PCR (Transnetyx) using primers specific to Hvcn1 intron 3 and pGT0Lxf ; results from the Transnetyx genotyping strategy were verified by triplex genomic PCR. .. Although quantitative RT-PCR confirmed that the RRN293 transgene was expressed at the expense of the wt Hvcn1 allele , we were unable to detect either β-galactosidase protein [using several different anti-β-galactosidase antibodies in Western blot analysis, immunocytochemistry (ICC) or immunohistochemistry (IHC)] or activity (using X-gal staining) in spleen or BMC from Hv1 −/− mice.

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    Transnetyx genotyping protocol
    Construction of the targeting vectors and generation of the corresponding mouse lines. A : A diagram illustrating the production of Chrna7 -IRES-tauGFP. In the first step, the IRES-tauGFP-FRT-neo-FRT cassette 1, including the HA epitope, was inserted in the 3′ untranslated region (UTR) of Chrna7 (Methods). The external hybridization probe position, PCR priming sites and Kpn I restriction sites are indicated. Numbered black boxes depict protein-coding exons. HA, hemagglutinin epitope; IRES, internal ribosomal entry site; FRT, recognition target sites of the FLP site-specific recombinase; neo, neomycin (kanamycin or G418 resistance); bovine tau polypeptide fusion to enhanced green fluorescent protein (tauGFP). B : The Chrna7 -IRES-Cre targeting vector was created by replacing tauGFP with the IRES-Cre-FRT-neo-FRT cassette 2. The external hybridization probe was as in (A). C : Southern blot showing Kpn I restriction fragment size upshift from 4.4 kb (WT, wild type) to 5.3 kb (TG, targeted allele) in the Chrna7 -IRES-tauGFP targeted ES cell DNA. NT, negative ES clones harboring non-homologous recombination. D : Chrna7 -IRES-Cre and Avr II restriction analysis producing an anticipated downshift from 7.6 kb (WT) to 5.2 kb. E : PCR <t>genotyping</t> of Chrna7 -IRES-tauGFP mice in which the FRT-neo-FRT selection cassette was removed (Methods). The PCR data demonstrates the absence of wild type allele in normally segregating homozygous (−/−) Chrna7 -IREStauGFP/IRES-tauGFP or Chrna7 -IRES-Cre animals.
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    Construction of the targeting vectors and generation of the corresponding mouse lines. A : A diagram illustrating the production of Chrna7 -IRES-tauGFP. In the first step, the IRES-tauGFP-FRT-neo-FRT cassette 1, including the HA epitope, was inserted in the 3′ untranslated region (UTR) of Chrna7 (Methods). The external hybridization probe position, PCR priming sites and Kpn I restriction sites are indicated. Numbered black boxes depict protein-coding exons. HA, hemagglutinin epitope; IRES, internal ribosomal entry site; FRT, recognition target sites of the FLP site-specific recombinase; neo, neomycin (kanamycin or G418 resistance); bovine tau polypeptide fusion to enhanced green fluorescent protein (tauGFP). B : The Chrna7 -IRES-Cre targeting vector was created by replacing tauGFP with the IRES-Cre-FRT-neo-FRT cassette 2. The external hybridization probe was as in (A). C : Southern blot showing Kpn I restriction fragment size upshift from 4.4 kb (WT, wild type) to 5.3 kb (TG, targeted allele) in the Chrna7 -IRES-tauGFP targeted ES cell DNA. NT, negative ES clones harboring non-homologous recombination. D : Chrna7 -IRES-Cre and Avr II restriction analysis producing an anticipated downshift from 7.6 kb (WT) to 5.2 kb. E : PCR genotyping of Chrna7 -IRES-tauGFP mice in which the FRT-neo-FRT selection cassette was removed (Methods). The PCR data demonstrates the absence of wild type allele in normally segregating homozygous (−/−) Chrna7 -IREStauGFP/IRES-tauGFP or Chrna7 -IRES-Cre animals.

    Journal: American Journal of Medical Genetics. Part a

    Article Title: Prenatal Ablation of Nicotinic Receptor alpha7 Cell Lineages Produces Lumbosacral Spina Bifida the Severity of Which is Modified by Choline and Nicotine Exposure

    doi: 10.1002/ajmg.a.35372

    Figure Lengend Snippet: Construction of the targeting vectors and generation of the corresponding mouse lines. A : A diagram illustrating the production of Chrna7 -IRES-tauGFP. In the first step, the IRES-tauGFP-FRT-neo-FRT cassette 1, including the HA epitope, was inserted in the 3′ untranslated region (UTR) of Chrna7 (Methods). The external hybridization probe position, PCR priming sites and Kpn I restriction sites are indicated. Numbered black boxes depict protein-coding exons. HA, hemagglutinin epitope; IRES, internal ribosomal entry site; FRT, recognition target sites of the FLP site-specific recombinase; neo, neomycin (kanamycin or G418 resistance); bovine tau polypeptide fusion to enhanced green fluorescent protein (tauGFP). B : The Chrna7 -IRES-Cre targeting vector was created by replacing tauGFP with the IRES-Cre-FRT-neo-FRT cassette 2. The external hybridization probe was as in (A). C : Southern blot showing Kpn I restriction fragment size upshift from 4.4 kb (WT, wild type) to 5.3 kb (TG, targeted allele) in the Chrna7 -IRES-tauGFP targeted ES cell DNA. NT, negative ES clones harboring non-homologous recombination. D : Chrna7 -IRES-Cre and Avr II restriction analysis producing an anticipated downshift from 7.6 kb (WT) to 5.2 kb. E : PCR genotyping of Chrna7 -IRES-tauGFP mice in which the FRT-neo-FRT selection cassette was removed (Methods). The PCR data demonstrates the absence of wild type allele in normally segregating homozygous (−/−) Chrna7 -IREStauGFP/IRES-tauGFP or Chrna7 -IRES-Cre animals.

    Article Snippet: An automated genotyping protocol for these alleles was developed by Transnetyx (using probes “Chrna7 -2 WT,” “eGFP,” and “CRE”).

    Techniques: Hybridization, Polymerase Chain Reaction, Plasmid Preparation, Southern Blot, Clone Assay, Homologous Recombination, Mouse Assay, Selection