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  • 92
    Thermo Fisher mouse brain complementary dna cdna library
    Mouse Brain Complementary Dna Cdna Library, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mouse brain complementary dna library
    Expression of the <t>D-AKAP2</t> protein in various mouse tissues. ( A ) Mouse tissue extracts were prepared as described in Experimental Procedures ). ( B ) Comparison of mouse and human full-length D-AKAP2. Human D-AKAP2 was in vitro translated by using a expression vector containing the <t>cDNA</t> as described in Experimental Procedures . It was then loaded onto SDS-PAGE along with a sample of mouse heart extract and probed with anti-D-AKAP2. M, mouse; H, human. The two arrows indicate the mobilities of the two full-length proteins. Lane 10 and M correspond to the same extract (*).
    Mouse Brain Complementary Dna Library, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse brain cdna
    Expression of the <t>D-AKAP2</t> protein in various mouse tissues. ( A ) Mouse tissue extracts were prepared as described in Experimental Procedures ). ( B ) Comparison of mouse and human full-length D-AKAP2. Human D-AKAP2 was in vitro translated by using a expression vector containing the <t>cDNA</t> as described in Experimental Procedures . It was then loaded onto SDS-PAGE along with a sample of mouse heart extract and probed with anti-D-AKAP2. M, mouse; H, human. The two arrows indicate the mobilities of the two full-length proteins. Lane 10 and M correspond to the same extract (*).
    Mouse Brain Cdna, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mouse brain marathon ready cdna
    The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time <t>PCR.</t> As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both <t>cDNA</t> fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.
    Mouse Brain Marathon Ready Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad complementary dna cdna
    The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time <t>PCR.</t> As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both <t>cDNA</t> fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.
    Complementary Dna Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 4319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse brain cdna
    The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time <t>PCR.</t> As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both <t>cDNA</t> fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.
    Mouse Brain Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zyagen mouse whole brain cdna
    The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time <t>PCR.</t> As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both <t>cDNA</t> fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.
    Mouse Whole Brain Cdna, supplied by Zyagen, used in various techniques. Bioz Stars score: 91/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mouse brain quick clone cdna
    The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time <t>PCR.</t> As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both <t>cDNA</t> fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.
    Mouse Brain Quick Clone Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of the D-AKAP2 protein in various mouse tissues. ( A ) Mouse tissue extracts were prepared as described in Experimental Procedures ). ( B ) Comparison of mouse and human full-length D-AKAP2. Human D-AKAP2 was in vitro translated by using a expression vector containing the cDNA as described in Experimental Procedures . It was then loaded onto SDS-PAGE along with a sample of mouse heart extract and probed with anti-D-AKAP2. M, mouse; H, human. The two arrows indicate the mobilities of the two full-length proteins. Lane 10 and M correspond to the same extract (*).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein

    doi: 10.1073/pnas.051633398

    Figure Lengend Snippet: Expression of the D-AKAP2 protein in various mouse tissues. ( A ) Mouse tissue extracts were prepared as described in Experimental Procedures ). ( B ) Comparison of mouse and human full-length D-AKAP2. Human D-AKAP2 was in vitro translated by using a expression vector containing the cDNA as described in Experimental Procedures . It was then loaded onto SDS-PAGE along with a sample of mouse heart extract and probed with anti-D-AKAP2. M, mouse; H, human. The two arrows indicate the mobilities of the two full-length proteins. Lane 10 and M correspond to the same extract (*).

    Article Snippet: Most of the missing N-terminal region of mouse D-AKAP2 was PCR amplified from mouse brain cDNA (CLONTECH) by using primers based on known mouse and human sequence, and random mouse cDNA sequences homologous to the human gene from GenBank.

    Techniques: Expressing, In Vitro, Plasmid Preparation, SDS Page

    Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Journal: British Journal of Pharmacology

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    doi: 10.1038/sj.bjp.0704671

    Figure Lengend Snippet: Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Article Snippet: These primers were used to obtain the full-length preproU-II complementary DNA (cDNA) clone from mouse brain cDNA template (Clonetech) by polymerase chain reaction (PCR).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Isolation, Derivative Assay

    Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Journal: British Journal of Pharmacology

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    doi: 10.1038/sj.bjp.0704671

    Figure Lengend Snippet: Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Article Snippet: These primers were used to obtain the full-length preproU-II complementary DNA (cDNA) clone from mouse brain cDNA template (Clonetech) by polymerase chain reaction (PCR).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Derivative Assay

    Identification of CTRP13 and its tissue expression profile. A , cloning of CTRP13 cDNA from overlapping EST sequences. The arrows indicate forward and reverse primers used to amplify the coding region of CTRP13 cDNA. B , agarose gel electrophoresis showing

    Journal: The Journal of Biological Chemistry

    Article Title: Metabolic Regulation by C1q/TNF-related Protein-13 (CTRP13): ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE AND SUPPRESSION OF FATTY ACID-INDUCED JNK SIGNALING*

    doi: 10.1074/jbc.M110.201087

    Figure Lengend Snippet: Identification of CTRP13 and its tissue expression profile. A , cloning of CTRP13 cDNA from overlapping EST sequences. The arrows indicate forward and reverse primers used to amplify the coding region of CTRP13 cDNA. B , agarose gel electrophoresis showing

    Article Snippet: CTRP13 cDNA was cloned from a mouse brain cDNA pool (Clontech) using the primer pair 5′-GGTGATGGTGCTTCTGCTGGTCATC-3′ and 5′-GATTCACTGACGTTAGCCATACG-3′ in a 35-cycle PCR using Platinum Pfx polymerase (Invitrogen) in the presence of 8% DMSO.

    Techniques: Expressing, Clone Assay, Agarose Gel Electrophoresis

    Sequence and structure of KyoT. (A) cDNA structure of KyoT. The coding regions are represented by thick lines. The cDNA fragments obtained from the mouse brain cDNA library are shown below the putative full-length cDNAs. The positions of primers used for RT-PCR are shown by arrows. E, Eco RI; H, Hin dIII; ATG, putative initiation codon; Stop, termination codon. (B) Nucleotide and deduced amino acid sequences of KyoT. Nucleotide sequences of coding and noncoding regions are shown as upper- and lowercase letters, respectively. The first 167 amino acids are shared between two alternatively spliced isoforms, KyoT1 and KyoT2. The amino acids removed by alternative splicing in KyoT2 are represented by dots. Four LIM domains are boxed, and the conserved cysteines and histidines within the LIM motif are shown as boldface letters. The 5′ ends of the fragments obtained by two-hybrid screening from mouse embryo (RAM14) and HeLa (hRAM8) cDNA libraries are shown by thin and thick arrows, respectively. (C) Genomic structure of KyoT. The functional gene is composed of at least five exons and spans at least 20 kbp. In KyoT2 mRNA, one of the exons is removed by alternative splicing. Most of the 5′ untranslated region was not cloned. Solid boxes, coding region; open boxes, untranslated region; ATG, first methionine of the coding region; TAA and TAG, termination codons of KyoT1 and KyoT2, respectively; E, Eco RI; B, Bam HI. (D) Simplified diagram showing the structures of the KyoT1 and KyoT2 proteins. The LIM domains are numbered from the N terminus.

    Journal: Molecular and Cellular Biology

    Article Title: LIM Protein KyoT2 Negatively Regulates Transcription by Association with the RBP-J DNA-Binding Protein

    doi:

    Figure Lengend Snippet: Sequence and structure of KyoT. (A) cDNA structure of KyoT. The coding regions are represented by thick lines. The cDNA fragments obtained from the mouse brain cDNA library are shown below the putative full-length cDNAs. The positions of primers used for RT-PCR are shown by arrows. E, Eco RI; H, Hin dIII; ATG, putative initiation codon; Stop, termination codon. (B) Nucleotide and deduced amino acid sequences of KyoT. Nucleotide sequences of coding and noncoding regions are shown as upper- and lowercase letters, respectively. The first 167 amino acids are shared between two alternatively spliced isoforms, KyoT1 and KyoT2. The amino acids removed by alternative splicing in KyoT2 are represented by dots. Four LIM domains are boxed, and the conserved cysteines and histidines within the LIM motif are shown as boldface letters. The 5′ ends of the fragments obtained by two-hybrid screening from mouse embryo (RAM14) and HeLa (hRAM8) cDNA libraries are shown by thin and thick arrows, respectively. (C) Genomic structure of KyoT. The functional gene is composed of at least five exons and spans at least 20 kbp. In KyoT2 mRNA, one of the exons is removed by alternative splicing. Most of the 5′ untranslated region was not cloned. Solid boxes, coding region; open boxes, untranslated region; ATG, first methionine of the coding region; TAA and TAG, termination codons of KyoT1 and KyoT2, respectively; E, Eco RI; B, Bam HI. (D) Simplified diagram showing the structures of the KyoT1 and KyoT2 proteins. The LIM domains are numbered from the N terminus.

    Article Snippet: KyoT1 and KyoT2 cDNAs were isolated from a mouse brain cDNA library (Clontech) by using the cDNA fragments recovered from the two-hybrid screen as probes, and both strands were sequenced with an Applied Biosystems automated sequencing apparatus.

    Techniques: Sequencing, cDNA Library Assay, Reverse Transcription Polymerase Chain Reaction, Two Hybrid Screening, Functional Assay, Clone Assay

    The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time PCR. As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both cDNA fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphatidylinositol 3-Phosphatase Myotubularin-related Protein 6 (MTMR6) Is Regulated by Small GTPase Rab1B in the Early Secretory and Autophagic Pathways *

    doi: 10.1074/jbc.M112.395087

    Figure Lengend Snippet: The mechanism of tubular omegasome formation. A , domain of DFCP1 responsible for tubular omegasome formation. Stable NRK cell lines expressing EGFP-DFCP1, EGFP-DFCP1 Δ-N, and EGFP-DFCP1 (domain mutant ( DM )) were starved for 120 min and observed with fluorescence microscopy. The diagrams show the structure of the constructs. CS , mutated FYVE domain. B , comparison of the amount of DFCP1 mRNA. The amount of DFCP1 mRNA was quantified by real time PCR. As our stable cell line expresses both EGFP-tagged recombinant mouse and endogenous rat DFCP1, PCR primers were designed to amplify both cDNA fragments (mean ± S.E. ( error bars ); n = 4). C , comparison of the amount of DFCP1 protein. Cell lysates were analyzed with anti-DFCP1 and EGFP antibodies by Western blotting. The numbers at the top indicate the amount of protein loaded, and the numbers on the right are molecular mass markers. Actin was used as a loading control. Bar, 10 μm.

    Article Snippet: Other cDNAs were amplified by PCR using mouse brain Marathon-Ready cDNA (Clontech) as the template.

    Techniques: Expressing, Mutagenesis, Fluorescence, Microscopy, Construct, Real-time Polymerase Chain Reaction, Stable Transfection, Recombinant, Polymerase Chain Reaction, Western Blot

    Generation of metabotropic glutamate receptors (mGluR1γ)-tagged knock-in (KI) mice and expression of mGluR1γ in mouse cerebellum. (A) Graphic representation of the CRISPR/Cas9 approach. To generate the mGluR1γ-tagged KI mice, single stranded oligodeoxynucleotide (ssODN) with the desired sequences was microinjected into wild type (WT) fertilized eggs together with Cas9 protein and crRNA/tracrRNA. (B) Targeting strategy to KI the HA and FLAG tags at the Grm1 locus. The double-strand break (DSB) was introduced by CRISPR/Cas9 and the 57-base HA/FLAG sequence flanked by 45-base homology arms was inserted into exon X of Grm1 . (C) Genomic DNA sequence around the KI site. The upper and lower sequences depict the genomic DNA and ssODN, respectively. The PAM sequence is shown in red, target sequence of crRNA in green and homology arms in blue. (D) Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from WT and mGluR1γ-tagged KI heterozygous (het) and homozygous (homo) mice. PCR fragments were digested with BamHI, and tag-insertion was confirmed by the presence of cleaved fragments by BamHI digestion. The expected size of BamHI-digested fragments is 389 and 9 bp in the WT allele, and 279, 167 and 9 bp in the KI allele. The largest fragment observed in the KI heterozygote lane may be derived from heteroduplex formation. (E) Expression of mGluR1 variants in WT and mGluR1γ-tagged KI mice. The diagram on the top schematically shows mGluR1 splice variants differing in their C-terminal domains (shown here in different colors). Protein extracts from the cerebella of WT and mGluR1γ-tagged homozygous KI mice were immunoblotted with antibodies against HA, FLAG, mGluR1 extracellular domain and β-actin. For anti-mGluR1, mGluR1α and mGluR1β are detected at around 150 and 100 kDa, respectively (right). No traces of mGluR1 variants were detected using HA (left) and FLAG (center) antibodies. (F) Reverse transcription PCR analysis of mGluR1 mRNA in cerebella of WT and mGluR1γ-tagged homozygous KI mice. The upper diagram shows alternative splicing patterns with locations of primers and expected sizes of PCR products. No detectable bands were observed at an expected size of the PCR product from mGluR1γ transcript. + and—indicate PCR analyses with and without reverse transcriptase, respectively. (G) ) could be amplified from human neocortex complementary DNA (cDNA), rat cerebellar cDNA, mouse whole brain and cerebellar cDNAs. Species-specific primers were used which gave rise to products of different lengths. (H) mGluR1γ transcripts could be amplified from all species by means of a nested PCR approach using as template gel-eluted bands between 200 bp and 400 bp from the first round of RT-PCRs.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: New Features on the Expression and Trafficking of mGluR1 Splice Variants Exposed by Two Novel Mutant Mouse Lines

    doi: 10.3389/fnmol.2018.00439

    Figure Lengend Snippet: Generation of metabotropic glutamate receptors (mGluR1γ)-tagged knock-in (KI) mice and expression of mGluR1γ in mouse cerebellum. (A) Graphic representation of the CRISPR/Cas9 approach. To generate the mGluR1γ-tagged KI mice, single stranded oligodeoxynucleotide (ssODN) with the desired sequences was microinjected into wild type (WT) fertilized eggs together with Cas9 protein and crRNA/tracrRNA. (B) Targeting strategy to KI the HA and FLAG tags at the Grm1 locus. The double-strand break (DSB) was introduced by CRISPR/Cas9 and the 57-base HA/FLAG sequence flanked by 45-base homology arms was inserted into exon X of Grm1 . (C) Genomic DNA sequence around the KI site. The upper and lower sequences depict the genomic DNA and ssODN, respectively. The PAM sequence is shown in red, target sequence of crRNA in green and homology arms in blue. (D) Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from WT and mGluR1γ-tagged KI heterozygous (het) and homozygous (homo) mice. PCR fragments were digested with BamHI, and tag-insertion was confirmed by the presence of cleaved fragments by BamHI digestion. The expected size of BamHI-digested fragments is 389 and 9 bp in the WT allele, and 279, 167 and 9 bp in the KI allele. The largest fragment observed in the KI heterozygote lane may be derived from heteroduplex formation. (E) Expression of mGluR1 variants in WT and mGluR1γ-tagged KI mice. The diagram on the top schematically shows mGluR1 splice variants differing in their C-terminal domains (shown here in different colors). Protein extracts from the cerebella of WT and mGluR1γ-tagged homozygous KI mice were immunoblotted with antibodies against HA, FLAG, mGluR1 extracellular domain and β-actin. For anti-mGluR1, mGluR1α and mGluR1β are detected at around 150 and 100 kDa, respectively (right). No traces of mGluR1 variants were detected using HA (left) and FLAG (center) antibodies. (F) Reverse transcription PCR analysis of mGluR1 mRNA in cerebella of WT and mGluR1γ-tagged homozygous KI mice. The upper diagram shows alternative splicing patterns with locations of primers and expected sizes of PCR products. No detectable bands were observed at an expected size of the PCR product from mGluR1γ transcript. + and—indicate PCR analyses with and without reverse transcriptase, respectively. (G) ) could be amplified from human neocortex complementary DNA (cDNA), rat cerebellar cDNA, mouse whole brain and cerebellar cDNAs. Species-specific primers were used which gave rise to products of different lengths. (H) mGluR1γ transcripts could be amplified from all species by means of a nested PCR approach using as template gel-eluted bands between 200 bp and 400 bp from the first round of RT-PCRs.

    Article Snippet: The subsequent PCR was performed using the following primers: forward 5′-GTGCCTTCACCACCTCTGAT-3′ reverse 5′-TGTAGTCGGATCCAGCGTAA-3′ For nested PCR, the following cDNAs were used as template for the PCRs: mouse cerebellar cDNA (Crepaldi et al., ), mouse whole brain Marathon-Ready cDNA (Clontech), rat cerebellar cDNA (Corti et al., ) and human cerebral cortex Marathon-Ready cDNA (Clontech).

    Techniques: Knock-In, Mouse Assay, Expressing, CRISPR, Sequencing, Polymerase Chain Reaction, Amplification, Derivative Assay, Nested PCR