To develop a SPIB expression vector, a FLAG tagged fragment of full length SPIB (FLAG‐SPIB) was sub‐cloned into the pCRII‐TOPO plasmid (Invitrogen in Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the vector was digested with EcoRI and transferred to the retrovirus vector MIGR1 (a kind gift from Dr W. S. Pear, University of Pennsylvania, Philadelphia, PA, USA), which constitutively expresses GFP, to generate MIGR1‐FLAG‐SPIB. The DNA sequence of FLAG‐SPIB cloned into MIGR1 was confirmed by sequence analysis. Transient transfection of the MIGR1 control vector (MOCK) and MIGR1‐FLAG‐SPIB into 293T cells was performed using the Effectene transfection reagent (Qiagen, Venlo, the Netherlands) according to the manufacturer's protocol. The stable transduction of SU‐DHL4 cells using MIGR1 vectors was performed using a retroviral infection system (Retro‐X Expression systems; Clontech in Takara Bio, Shiga, Japan). In brief, the MIGR1 vector and envelope vector (pVSV‐G) were co‐transfected into the GP2‐293 packaging cell line using the FuGENE6 transfection reagent (Promega, Fitchburg, WI, USA). Two to 4 days later, retrovirus was harvested from the culture supernatant and applied to SU‐DHL4 cell lines. SU‐DHL4 cells transduced with MIGR1 vector were sorted for GFP expression using a FACSAria II (Becton‐Dickinson [BD], Franklin Lakes, NJ, USA).
Pcrii topo plasmid
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PCRII-TOPO plasmid is a cloning vector used in molecular biology. It is designed for the direct cloning of Taq polymerase-amplified PCR products. The plasmid contains a TOPO site that enables the direct insertion of PCR products without the need for ligation.
Automatically generated - may contain errors
Lab products found in correlation
Dig rna labeling mix, by Roche (2 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Facsaria 2, by BD (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Gel dna recovery kit, by Zymo Research (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
T7 or sp6 rna polymerase, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Genelute mammalian genomic dna miniprep kit, by Merck Group (1 mentions)
Cell culture insert, by Greiner (1 mentions)
Nucleospin extract 2, by Macherey-Nagel (1 mentions)
Bx61 microscope, by Adobe (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rhodamine labeled dolichos biflorus agglutinin, by Vector Laboratories (1 mentions)
Superscript 3 cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
12 protocols using pcrii topo plasmid
1
Generating SPIB Expression Vector
2
Isolation of Sigmar1 splice variants
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The first-strand cDNAs from the brain of CD-1 mice were used as a template to isolate Sigmar1 splice variants using Platinum Taq DNA polymerase with a sense primer (pSE1:5’-GTA GGA TCC ATG CCG TGG GCC GCG GG-3’ ) and an antisense primer (pAN1: 5’-GCA TCT CTG TGT CTC ATT TGC TTC CC-3’ ; or pAN2: GTT GAA TTC GAG AGA TGG ATG TGG TCC TGC CGC-3’ ). The PCR fragments with different sizes were excised and purified from an agorase gel using a Gel DNA Recovery Kit (ZYMO Research) and subcloned into a pCRII-TOPO plasmid (Invitrogen). The subcloned PCR fragments were sequenced using appropriate primers.
3
Cloning of paHSPC152 and paTYR Plasmids
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PTER+ plasmid was used for all cloning [18 (link)], except for paTYR sense, where pcDNA3 plasmid was used. Genomic fragments matching the paRNAs were amplified by PCR, using the PhusionTM master mix (cat# F-531S, Finnzymes OY, 02150 Espoo, Finland) with 10 pmol of the following primers:
HSPC152 F-5′-CGACGGTGTTAGGCGC 3′
HSPC152 R-5′-CGGGTACCTGGAGGCG 3′
TYR F-5′-CATTTGCAAGGTCAAATCATC 3′
TYR R-5′-AGTACAAAACAGCCAGGAGC 3′
4
DNA Nicking Assay with Gelatin Hydrolysates
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DNA nicking assay was performed using pCRII TOPO plasmid (invitrogen). A mixture of 10 μL of gelatin hydrolysates at the concentration of 2 mg/mL and plasmid DNA (0.5 μg/well) were incubated for 10 min at room temperature followed by the addition of 10 μL of Fenton's reagent (30 mM H2O2, 50 μM L-ascorbic acid, and 80 μM FeCl3). The mixture was then incubated for 5 min at 37°C. The DNA was analysed on 1% (w/v) agarose gel using ethidium bromide staining.
5
Cloning and Probing Zebrafish Genes
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The sequences of zebrafish PIH genes and DNAH genes were subcloned into pCRII-TOPO plasmid (Invitrogen). From the constructed plasmids, RNA probes were synthesized using SP6 or T7 RNA polymerase (Roche) with DIG RNA Labeling Mix (Roche). RNAs were purified using RNeasy Mini Kit (Qiagen). Sequences of primers used in the construction of plasmids are summarized in Table 2 .
6
Probe Design for In Situ Hybridization
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A list of all transcripts which were used as targets for in situ hybridization probes is given in Table 1 . For all probes which have been described previously, references are given in Supplementary Table S1 . Templates for all other probes were PCR amplified from cDNA and subcloned into pCRII-TOPO plasmid (Invitrogen) as has been described before (Schredelseker and Driever, 2018 (link)). Primer sequences are shown in Supplementary Table S1 .
7
Genomic DNA Isolation and PCR Amplification
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The genomic DNA was isolated using GenElute™ Mammalian Genomic DNA Miniprep Kit (G1N70, Sigma). The PCR fragments (581 bp) were amplified using Fwd and Rv primers (Supplemental Table S1 ) with the following conditions: denaturing for 30 sec at 95 °C, followed by 32 cycles of 10 sec at 95 °C, 30 sec at 58 °C, and 30 sec at 72 °C, followed by 5 min of final extension at 72 °C. The amplicons were then cloned into the pCRII-TOPO plasmid (Invitrogen, Merelbeke, Belgium) and sequenced with primers M13-Fwd and M13-Rev (Supplemental Table S1 ).
8
In situ hybridization of insect pheromone-binding genes
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For slnlg4-yll and slnrxI, cDNA fragments of respectively 748 bp and 565 bp were amplified by PCR using specific primers (Table S1), purified (Nucleospin Extract II, Macherey-Nagel) and cloned into pCRII-TOPO plasmid (Invitrogen), used as template for in vitro transcription to generate DIG-labeled RNA sense and antisense probes. The Pheromone-Binding protein SlPBP1 (GenBank accession number EF396284), whose transcripts are highly expressed in male antennae, was used as positive control. Antennae from three-day-old male moths were embedded in Tissue Tek 186 medium TM compound (CellPath, Newtown Powys, UK). Cryosections (7 µm) were set in cell culture insert (Greiner Bio-one, Monroe, USA). Hybridization was conducted as described previously (Durand et al. 2010a ). Pictures were acquired (Olympus BX61 microscope, ImagePro software) and digitalized using Adobe Photoshop ® 7.0 (Adobe, USA).
9
In situ hybridization of Plag1 in mouse epididymis
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Epididymal sections from 7-week-old Plag1 WT and KO male mice (n = 5 per genotype) were made as described above for X-gal staining. In situ hybridization was performed as previously described10 (link) with the following alterations: riboprobes were transcribed from 1 μg of linearized pCRII-TOPO plasmid (Thermo Fisher Scientific, Waltham, MA, USA) containing a 752-bp insert (503–1254 bp of the mouse Plag1 coding sequence, GenBank accession number NM_019969.3) in digoxigenin (DIG) RNA Labeling Mix and 40 U SP6 or T7 RNA polymerase (Roche Diagnostics, Risch-RotKreuz, Switzerland). The slides were incubated overnight in the hybridization buffer containing 800 ng ml−1 antisense or sense DIG-labeled probe at 66°C. Coverslips were removed and the slides were washed two times for 5 min in 0.2× saline sodium citrate at room temperature, followed by three 5-min washes in PBS with 0.1% (v/v) Triton X-100 (Sigma-Aldrich). The protocol continued as described before.10 (link) As an additional negative control, epididymis sections from a Plag1 KO male were included to ensure there was no nonspecific probe binding. The sections were imaged as described above.
10
Multimodal Analysis of LRH-1 Expression
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Immunofluorescence (IF) and RNA in situ hybridization were performed on 5 μm cryosections using standard procedures. DIG-labeled (Roche) riboprobes were generated from pCRII-TOPO plasmid (ThermoFisher Sci) with mLRH-1 cDNA corresponding to bases 595–1683. Antibodies against hLRH-1 (1:200, Sigma HPA005455), FLAG (1:300, Sigma F7425), cleaved Caspase-3 (1:1000 (WB) and 1:400 (IF), Cell Signaling 5A1E), CD-44 (1:500, Tonbo 70-0441), lysozyme (1:200, DAKO EC 3.2.1.17) and MUC-2 (1:300, Santa Cruz Biotechnology sc-15334) were used with Alexa Fluor-conjugated secondary antibodies 1:300 (Millipore, Invitrogen). For Goblet staining, Rhodamine-labeled Dolichos Biflorus Agglutinin (Vector Labs) was used at 1:200 dilution.
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