The [35S]IA-2 tracer was obtained by in vitro transcription/translation of the cDNA coding for human IA-2iccloned into pSP64 vector (Promega, Madison, WI, USA), using a rabbit reticulocyte lysate system (Promega, Madison, WI, USA) in the presence of [35S]-methionine (New England, Nuclear, Boston, MA, USA), according to the manufacturer’s instructions. Translation products were diluted in radioimmunoassay (RIA) buffer (0.02 M Tris-HCl, 0.15 M NaCl, 0.1 % v/v Tween 20, pH 7.4) and applied to a PD10 column (Pharmacia-LKB Biotechnology, Uppsala, Sweden) in order to remove free [35S]-methionine. Typically, the percentage of incorporation of [35S]-methionine to the protein by this method was 5–7 %, yielding about 5–7×106cpm of labelled protein. The tracer was stored in aliquots at -40 °C, and had a shelf life of 5 weeks.
Psp64 vector
Manufactured by Promega
Sourced in United States
The PSP64 vector is a plasmid vector designed for the expression of proteins in eukaryotic cells. The vector contains a multiple cloning site for the insertion of a gene of interest, as well as regulatory elements necessary for transcription and translation in eukaryotic cells. The vector also includes elements for the selection of transformed cells, such as an antibiotic resistance gene.
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Lab products found in correlation
Sp6 rna polymerase, by Promega (7 mentions)
Riboprobe, by Promega (7 mentions)
Ecori, by New England Biolabs (7 mentions)
Ex taq polymerase, by Takara Bio (2 mentions)
Rabbit reticulocyte lysate system, by Promega (1 mentions)
Linear polyethylenimine, by Polysciences (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Gigaprep, by Qiagen (1 mentions)
Pdr274 vector, by Addgene (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Opti mem 1, by Merck Group (1 mentions)
16 protocols using psp64 vector
1
Radiolabeling IA-2 Autoantigen for Immunoassay
2
Synthesis of Capped mRNA Transcripts
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Example 12
A gene of interest is cloned into the multiple cloning site of the pSP64 vector (Promega) using standard molecular biology methods. The vector is digested with EcoRI (NEB) to generate a linearized dsDNA vector containing the SP6 promoter, gene of interest, and 30 nucleotide long poly-A tail. mRNA is synthesized by reaction with SP6 RNA polymerase (Promega) according to manufacturer's instructions, including recommended concentrations of 5′ cap analog (ARCA) to synthesize capped mRNA transcript. The reaction mixture is then treated with DNAse to digest the template vector (Riboprobe from Promega) and the mRNA is purified using the EZNA MicroElute RNA Clean-Up kit (Omega).
3
Generation of mASIC1a Concatemeric Constructs
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Mouse ASIC1a concatemeric constructs were generated as described previously (Lynagh et al., 2017 (link)). Briefly, the concatemeric mASIC1a constructs were generated by cloning out the mASIC1a insert from the pSP64 vector using forward and reverse primers containing additional HindIII and SalI, SalI and BamHI, or BamHI and SacI sequences, respectively (Figure 4—figure supplement 1 ). This generated three distinct inserts, a, b, and c, which were gel-purified using the Gel/PCR DNA fragment kit (Geneaid). The inserts were digested with their respective restriction enzymes – insert a: HindIII and SalI; insert b: SalI and BamHI; and insert c: BamHI and SacI. Insert a was ligated into the empty pSP64 vector (Promega) double-digested with HindIII and SalI. The resulting vector was then double-digested with SalI and BamHI to insert segment b in similar way. The a-b plasmid was then double-digested using BamHI and SacI and segment c was ligated into it. This yielded the final ‘a-b-c’ mASIC1a concatemer. From this construct, WT inserts were replaced by mASIC1a inserts containing amino acid substitutions generated using the same primers and restriction sites. Full concatemer sequences were confirmed using primers recognizing the unique restriction sites.
4
Recombinant Protein and RNA Synthesis
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The Roquin ROQ domain used in this study was expressed from the pETTrx1a vector, obtained from Gunther Stier (EMBL); its cloning has been described before (45 (link)). For protein purification of the RNA recognition motifs of AUF1 (AUF1_RRM2 and AUF1_RRM1-2), coding sequences were generated by PCR amplification using pCMV-hnRNP D37 as a template (47 (link)) and introduced into XbaI and XhoI sites of an His6- and thioredoxin-tag encoding plasmid based on pETTrx1a, i.e. including a TEV (tobacco etch virus) site for proteolytic removal of all tags.
For RNA synthesis, CDE1, CDE2, CDE1GG, CDE2GG and CDE1-2 (tandem) sequences together with the T7 promoter were generated by hybridization of complementary oligonucleotides and introduced into the NcoI and HindIII sites of an HDV ribozyme encoding plasmid based on the pSP64 vector (Promega). RNAs were transcribed as HDV ribozyme fusions to obtain uniform 3′ ends.
For the luciferase reporter system, 3′-UTR variants were generated by hybridization of complementary oligonucleotides and introduced downstream of the firefly luciferase open reading frame into the multiple cloning site of pDLP (48 (link)) using NotI and HindIII restriction sites.
All protein and RNA sequences are summarized inSupplementary Tables S1 and S2 .
For RNA synthesis, CDE1, CDE2, CDE1GG, CDE2GG and CDE1-2 (tandem) sequences together with the T7 promoter were generated by hybridization of complementary oligonucleotides and introduced into the NcoI and HindIII sites of an HDV ribozyme encoding plasmid based on the pSP64 vector (Promega). RNAs were transcribed as HDV ribozyme fusions to obtain uniform 3′ ends.
For the luciferase reporter system, 3′-UTR variants were generated by hybridization of complementary oligonucleotides and introduced downstream of the firefly luciferase open reading frame into the multiple cloning site of pDLP (48 (link)) using NotI and HindIII restriction sites.
All protein and RNA sequences are summarized in
5
Synthesis of Capped mRNA from Cloned Gene
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Example 2
A gene of interest is cloned into the multiple cloning site of the pSP64 vector (Promega) using standard molecular biology methods. The vector is digested with EcoRI (NEB) to generate a linearized dsDNA vector containing the SP6 promoter, gene of interest, and 30 nucleotide long poly-A tail. mRNA is synthesized by reaction with SP6 RNA polymerase (Promega) according to manufacturer's instructions, including recommended concentrations of 5′ cap analog (ARCA) to synthesize capped mRNA transcript. The reaction mixture is then treated with DNAse to digest the template vector (Riboprobe from Promega) and the mRNA is purified using the EZNA MicroElute RNA Clean-Up kit (Omega).
6
Uniform RNA Synthesis via HDV Ribozyme
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For RNA synthesis, UCP3 wt, MUTI, MUTII, MUTI/II, R1, CDEImut, CDEIImut, CDEI/IImut sequences together with the T7 promoter were generated by hybridization and introduced into the NcoI and HindIII sites of an HDV ribozyme encoding plasmid based on the pSP64 vector (Promega). RNAs were transcribed as HDV ribozyme fusions to obtain uniform 3′ ends. Required sequences were obtained by hybridization of complementary oligonucleotides and phosphorylation of restriction sites.
7
BLV Vaccine Production Protocol
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The BLV vaccine is a recombinant between two previously reported modified strains displaying an attenuated phenotype (10 (link), 11 (link)). Briefly, the vaccine strain (pBLV6073DX) has a point mutation in the transmembrane protein gene (T>G at nucleotide 6073) and a partial deletion of the R3-G4 sequences (between positions 6614 and 6997) (Figure 1A
8
Synthesis of Capped mRNA Transcripts
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Example 12
A gene of interest is cloned into the multiple cloning site of the pSP64 vector (Promega) using standard molecular biology methods. The vector is digested with EcoRI (NEB) to generate a linearized dsDNA vector containing the SP6 promoter, gene of interest, and 30 nucleotide long poly-A tail. mRNA is synthesized by reaction with SP6 RNA polymerase (Promega) according to manufacturer's instructions, including recommended concentrations of 5′ cap analog (ARCA) to synthesize capped mRNA transcript. The reaction mixture is then treated with DNAse to digest the template vector (Riboprobe from Promega) and the mRNA is purified using the EZNA MicroElute RNA Clean-Up kit (Omega).
9
Synthesis of Capped mRNA with SP6 Polymerase
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Example 2
A gene of interest is cloned into the multiple cloning site of the pSP64 vector (Promega) using standard molecular biology methods. The vector is digested with EcoRI (NEB) to generate a linearized dsDNA vector containing the SP6 promoter, gene of interest, and 30 nucleotide long poly-A tail. mRNA is synthesized by reaction with SP6 RNA polymerase (Promega) according to manufacturer's instructions, including recommended concentrations of 5′ cap analog (ARCA) to synthesize capped mRNA transcript. The reaction mixture is then treated with DNAse to digest the template vector (Riboprobe from Promega) and the mRNA is purified using the EZNA MicroElute RNA Clean-Up kit (Omega).
10
In Vitro Transcription of CD133 Protein
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Plasmid constructs have been described previously.15 (link),16 (link),26 (link) Briefly, for in vitro transcription, the plasmids were cloned with pSP64 vector (Promega). A TRP-2 signal sequence fragment and TM/cyto domain were amplified from TRP-2 cDNA by using PCR (Ex Taq polymerase; Takara Bio). The PCR products were cloned as a HindIII-PstI signal sequence fragment and a BamHI-SmaI TM/cyto domain fragment into pSP64 (Figure S3 ) to allow in vitro transcription under the control of an SP6 promoter to transport the CD133 protein efficiently to MHC class II compartments for eventual cross-presentation by both classes I and II on DCs in a cognate manner.
The total RNAs of the BTSC5 and GL261 neurosphere cells were collected for reverse transcriptase PCR by using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Primers used can be found inSupplemental Materials and Methods .
The total RNAs of the BTSC5 and GL261 neurosphere cells were collected for reverse transcriptase PCR by using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Primers used can be found in
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