All cysteine substitution mutants and the GS1 and GS2 constructs were generated by strand overlap extension PCR (4 (link)) using a cDNA encoding DDR1 C287S as a template. DDR1 C287S and the JM deletion mutants were created by the same method, using a cDNA encoding wild-type human DDR1b as template. The primer sequences used to generate the mutations are available on request. PCR products containing the relevant mutations were cut with SacI and XhoI and subcloned into a pGEM-3Z (Promega)-based vector encoding full-length DDR1b, after the corresponding wild-type sequence was removed. cDNAs encoding mutant DDR1b were cloned into the mammalian expression vector pRK5 (BD Biosciences). All PCR-derived DNA constructs were verified by DNA sequencing.
Pgem 3z
Manufactured by Promega
Sourced in United States
pGEM-3Z is a small, high-copy-number plasmid vector that can be used for cloning, sequencing, and in vitro transcription. It contains multiple cloning sites, a lacZ gene for blue/white screening, and T7 and SP6 RNA polymerase promoters flanking the multiple cloning site.
Automatically generated - may contain errors
Lab products found in correlation
Td 20 20 luminometer, by Turner Designs (2 mentions)
Pronase, by Boehringer Ingelheim (1 mentions)
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
Megashortscript t7 kit, by Thermo Fisher Scientific (1 mentions)
Amplicap maxt7 high yield message maker kit, by Illumina (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Mastercycler nexus, by Eppendorf (1 mentions)
7 protocols using pgem 3z
1
DDR1 Mutant Generation by PCR
2
Quantification of LacZ Reporter Activity
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Chemiluminescent quantification of the reporter pRAREhsplacZ plasmid (Rossant et al., 1991 (link)) activity was performed using ß-gal Juice Plus (PJK, Germany) as previously described (Yelin et al., 2005 (link)). Chemiluminescence activity was measured on a TD-20/20 Luminometer (Turner Designs). LacZ RNA was prepared from a clone containing a nuclear localization signal (pSP6nuc ß-gal) in pGEM-3Z (Promega). The staining of embryos for ß-galactosidase activity was performed with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal).
3
In Situ Hybridization for Cytokine RNA Detection
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After linearisation of plasmids (pGEM-3Z, Promega, Madison, Wisconsin, USA) containing specific sequences of the genes for hIL-1beta (R&D Systems, Minneapolis, USA) and hIL-1R type 1 and type 2 (kindly provided by Immunex, Seattle, WA, USA), 35S-labeled run-off anti-sense and sense (control-) transcripts were generated using Sp6 and T7 RNA polymerases (Gibco BRL). ISH for the detection of RNA transcripts was performed as previously described [2 (link)]. In brief, dewaxed and rehydrated paraffin sections were exposed to 0.2 N HCL and 0.125 mg/ml pronase (Boehringer, Mannheim, Germany) followed by acetylation with 0.1 M triethanolamine pH 8.0/0.25% (v/v) acetic anhydride and dehydration through graded ethanols. Slides were hybridized to 2–4 x 105 cpm of labeled probes overnight at 54°C. Washing and autoradiography was performed as described [2 (link)]. All sections were processed in parallel using the same batches of reagents and probes. The incubation of sections with Micrococcus nuclease (Boehringer Mannheim, Mannheim, Germany) prior to in situ hybridization resulted in the extinction of the specific autoradiographic signal, establishing that RNA sequences were the targets of the hybridization procedure. ISH signals were semiquantitated by counting the proportion of positive HRS cells and estimating the density of silver grains as the correlate for the transcript levels.
4
In vitro gRNA Synthesis and CRIS-PITCh Vector Construction
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In vitro transcribed gRNAs were prepared according to a previous report (Aida et al., 2015 (link)). Briefly, template DNA fragments were generated using PCR amplification from CRISPR-Cas9 vectors with primers containing a T7 promoter sequence according to a previous protocol (Nakagawa et al., 2016 (link)). Subsequently, the gRNAs were synthesized using a MEGAshortscript T7 Kit (Life Technologies, Carlsbad, CA, USA), and then purified with a MEGAclear Kit (Life Technologies). For the CRIS-PITCh (v2) vector, a genomic region containing Spp1 exons 3 and 4 was amplified from mouse genomic DNA and cloned into pGEM-3Z (Promega, Tokyo, Japan). Subsequently, base substitutions were introduced by site-directed mutagenesis. The two PITCh-gRNA target sites were then added to flank the microhomology sequences. The ssODNs were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The sequences of oligonucleotides for gRNA templates, primers, and ssODNs are listed in Table S3 . The recombinant Cas9 protein was obtained from New England Biolabs Japan (Cas9 Nuclease NLS, Streptococcus pyogenes; Tokyo, Japan) or Integrated DNA Technologies Japan (Alt-R™ S.p. Cas9 Nuclease 3NLS; Tokyo, Japan).
5
Cloning eNpHR3.0 DNA into Oocyte Vectors
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eNpHR3.0 DNA was cloned into oocyte expression vectors, based on the plasmid pGEMHE 22, a derivative of pGEM3z (Promega). NheI-linearized plasmid DNA was used for the in vitro generation of cRNA with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies).
6
Chemiluminescent Quantification of lacZ Reporter
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Chemiluminescent quantification of the reporter pRAREhsplacZ plasmid (Rossant et al., 1991 (link)) activity was performed using ß-gal Juice Plus (PJK, Germany) as previously described (Yelin et al., 2005 (link)). Chemiluminescence activity was measured on a TD-20/20 Luminometer (Turner Designs). LacZ RNA was prepared from a clone containing a nuclear localization signal (pSP6nuc ß-gal) in pGEM-3Z (Promega). The staining of embryos for ß-galactosidase activity was performed with 5-bromo-4-chloro-3-indolyl-β-d -galactopyranoside (Xgal).
7
Bacterial 16S rRNA Gene Amplification
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The 16 rRNA gene, considered as a molecular signature to identify members of bacterial communities, is conserved among prokaryotes with specific variable regions.
The 16S rRNA gene was amplified by PCR using universal primers 27F (5′-AGAGTTT GATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).13 PCRs were performed in a final volume of 50 µL, containing 2 µL of DNA (50 ng/µL), 2 µL of 1 × buffer, 3 mM MgCl2, 0.2 mM each dNTP, 1 U Taq Polymerase (Fermentas, Waltham, MA, USA), 0.4 μM direct and reverse primers, and deionized water. PCR amplification was performed using a thermocycler (Mastercycler Nexus-Eppendorf) as follows: denaturation at 95° C for five minutes, followed by 30 cycles of denaturation at 94° C for 45 seconds, alignment at 56° C for 45 seconds, and extension at 72° C for 90 seconds, and a final extension of 72° C for 10 minutes. Products were separated on 1.5% agarose gels in 1 × TBE buffer stained with ethidium bromide (0.5 mg/mL) and visualized under UV light. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instructions. The purified PCR products were sequenced (ABI PRISM 3500 Automatic Sequencer) using the vector pGEM®-3Z (Promega, Madison, WI, USA) as a positive control.
The 16S rRNA gene was amplified by PCR using universal primers 27F (5′-AGAGTTT GATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).13 PCRs were performed in a final volume of 50 µL, containing 2 µL of DNA (50 ng/µL), 2 µL of 1 × buffer, 3 mM MgCl2, 0.2 mM each dNTP, 1 U Taq Polymerase (Fermentas, Waltham, MA, USA), 0.4 μM direct and reverse primers, and deionized water. PCR amplification was performed using a thermocycler (Mastercycler Nexus-Eppendorf) as follows: denaturation at 95° C for five minutes, followed by 30 cycles of denaturation at 94° C for 45 seconds, alignment at 56° C for 45 seconds, and extension at 72° C for 90 seconds, and a final extension of 72° C for 10 minutes. Products were separated on 1.5% agarose gels in 1 × TBE buffer stained with ethidium bromide (0.5 mg/mL) and visualized under UV light. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instructions. The purified PCR products were sequenced (ABI PRISM 3500 Automatic Sequencer) using the vector pGEM®-3Z (Promega, Madison, WI, USA) as a positive control.
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