CRMP2 mRNA was extracted from mature 3T3-L1 adipocytes and reverse-transcribed into cDNA. The CRMP2 gene fragments were amplified by myc taq-containing primers 5′-ATGTCTTATCAGGGGAAGAAAAATATTCCA-3′ and 5′-TTACAGATCTTCTTCAGAAATAAGTTTTTGTTCGCCCAGGCTGGTGATGT-3′) and subcloned into pTARGETTM vector (Promega, Madison, WI, USA)) to generate CRMP2-carrying pTARGETTM vector (pTARGETTM-CRMP2-Myc). For CRMP2 overexpression, cells were transfected with 1 μg of pTARGETTM-CRMP2-Myc using PolyJetTM reagent (SignaGen Laboratories, Rockville, MD, USA) on day 2 and day 4, respectively, to analyze the effects of CRMP2 overexpression on the entire adipogenesis and lipid accumulation in the late adipogenic phase. For siRNA transfection, the pre-adipocytes were transfected with either 100 nM scramble siRNA or CRMP2 siRNA cocktail by DharmaFECT 1 reagent on day -2 according to the manufacturer’s instruction. The cells were incubated in medium containing the siRNA-DharmaconFECT1 complex for 16 h, and induced into differentiation after being incubated for another 24 h with fresh DMEM supplemented with 10% calf serum.
Ptarget vector
Manufactured by Promega
Sourced in United States
The PTarget vector is a versatile plasmid designed for protein expression in a variety of host systems. It features a T7 promoter for high-level recombinant protein production and multiple cloning sites for convenient gene insertion. The vector backbone provides antibiotic resistance and options for affinity tag incorporation to facilitate protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Geneart site directed mutagenesis system, by Thermo Fisher Scientific (5 mentions)
Onetaq 2 master mix with standard buffer, by New England Biolabs (4 mentions)
Sequencher 4, by Gene Codes (4 mentions)
Polyjet reagent, by SignaGen (1 mentions)
Pgl3 control dual luciferase mirna target expression vector, by Promega (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Prk5 ha ubiquitin wt plasmid, by Addgene (1 mentions)
Pgex 4t 1 vector, by Addgene (1 mentions)
Pcr2.1 topo cloning vector, by Thermo Fisher Scientific (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
Roswell park memorial institute (rpmi), by Nacalai Tesque (1 mentions)
Thp 1, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
G1471, by Promega (1 mentions)
Quikchange site directed mutagenesis kit, by Thermo Fisher Scientific (1 mentions)
Quikchange lightning mutagenesis kit, by Agilent Technologies (1 mentions)
Flag dgcr8, by Addgene (1 mentions)
Longamp taq dna polymerase, by New England Biolabs (1 mentions)
17 protocols using ptarget vector
1
CRMP2 Overexpression in Adipogenesis
2
Overexpression and Luciferase Assays of LINC00052, NTRK3, and miRNAs
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LINC00052 fragment was obtained by PCR, then the fragment was cloned into pcDNA3.1(+) vector and named as pcDNA3.1-LINC00052. The over expression vector of NTRK3 (pCMV-Sport6-NTRK3) was created by cloning the NTRK3 coding sequence into pCMV-Sport6 vector with the Kpn I/Xho I sites. The miR-128 and miR-485-3p fragments were amplified by PCR using the genomic DNA of SMMC7721 cells as a template. Then the amplified fragments were cloned into pTargetTM vector (Promega), named pTarget-128 and pTarget-485-3p respectively. The wild-type NTRK3 3′-UTR was amplified by PCR from genomic DNA as a template, and the PCR product was subcloned into pGL3-Control dual-luciferase miRNA target expression vector (Promega) immediately downstream of the luciferase gene, named pGL3-NTRK3 3′-UTR. All vectors constructed were confirmed by DNA sequencing. All primers are listed in Table 1 .
3
Oligonucleotide Protocols for Sequencing, Cloning, and Splicing
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Oligonucleotides were purchased from Sigma (St Louis, MO, USA). They were used in sequencing, cloning, and splicing assay (see below) experiments, and their sequence is available on request.
The pTarge T vector was purchased from Promega (Madison, WI, USA). The α-globin-fibronectin hybrid vector (modified pBS-KS) [32 (link)] was a kind gift of Dr. Emanuele Buratti (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy).
The pT
4
Generation of Lentiviral E3 Ligase Constructs
5
Establishing HPV16 E6/E7 Expressing Cell Lines
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pTARGET/E6E7 was prepared as previously described [14 (link)]. In short, E6/E7 derived from HPV16 were amplified by PCR from total RNA isolated from CaSki cell lines using a following primer set: 5’-GCG GCC GCC ACC ATG TTT CAG GAC CAC AG-3’ (Forward) and 5’-AGG CGG CCG CGA TTA TGG TTT CTG AGA ACA-3’ (Reverse). The PCR products were inserted into pCR2.1-TOPO cloning vector (Invitrogen, USA) and digested with EcoRI. The excised E6/E7 vector was ligated into the pTARGET vector (Promega) to construct pTARGET/E6E7. We transfected the pTARGET/E6E7 construct (5 μg/well) into primary HDPCs using the OmicsFect in vitro transfection reagent (OmicsBio, Taiwan). Twenty-four hours after transfection, 500 μg/ml of G-418 was added to the wells in the transfection group and incubated for an additional 24 h. Only G-418 selected cells were used.
6
Establishing LVRN-Transfected Swan71 Cells
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The human acute monocytic leukemia cell line THP-1 (RRID: CVCL_0006) was purchased from ATCC. To activate THP-1 cells, we added Phorbol12-myristate13-acetate (PMA, Sigma-Aldrich) and cultured them for 48h. Swan71 (RRID: CVCL_D855) was gifted by Prof. Mor G, which is human telomerase reverse transcriptase-transfected immortalized first-trimester trophoblast cell line.18 (link) Both cell lines were authenticated by STR profiling.
To establish LVRN-transfected Swan71 cells (Swan71_LVRN), hLVRN-His cDNA was inserted into pTargeT vector (Promega). Then, r-LVRN-His/pTargeT (500 ng) was cleaved and linearized with Psp1406I and transfected into Swan71 cells using FuGeneHD (Promega). After 48 h of gene transfer, a stable expression strain was established and isolated using a medium containing 500 μg/mL G418. For controls, the non-inserted pTargeT vector was transfected into Swan71 cells (Swan71_NEO). Cell lines were maintained at 37°C under 5% CO2 in RPMI (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS (Sigma-Aldrich), 100 g/mL streptomycin, and 100 IU/mL penicillin. Mycoplasma infections were detected regularly, and all experiments were performed under mycoplasma-free conditions.
To establish LVRN-transfected Swan71 cells (Swan71_LVRN), hLVRN-His cDNA was inserted into pTargeT vector (Promega). Then, r-LVRN-His/pTargeT (500 ng) was cleaved and linearized with Psp1406I and transfected into Swan71 cells using FuGeneHD (Promega). After 48 h of gene transfer, a stable expression strain was established and isolated using a medium containing 500 μg/mL G418. For controls, the non-inserted pTargeT vector was transfected into Swan71 cells (Swan71_NEO). Cell lines were maintained at 37°C under 5% CO2 in RPMI (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS (Sigma-Aldrich), 100 g/mL streptomycin, and 100 IU/mL penicillin. Mycoplasma infections were detected regularly, and all experiments were performed under mycoplasma-free conditions.
7
Cloning and Mutating Human OCT2 Promoter
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The part of the sequence of the human OCT2 promoter containing the predicted NF-kB binding site was cloned from a commercially available human genomic library (G1471, Promega, Fitchburg, WI, USA) using the forward primer 5'-cccgacggctcttgttgttggttg-3' and the reverse primer 5'-gcgcgaaggtagccgagagcaga-3'. A 689-bp amplicon corresponding to the−586 / +103 fragment flanking the transcription starting site of the SLC22A2 gene was isolated and blunt ligated into the pTargeT vector (A1410, Promega, Madison, WI, USA). The insert was then subcloned in pMCS-Luc vector (16146, ThermoFisher scientific, Waltham, MA, USA) by digestion with NheI and Smal restriction enzymes. Mutants were generated using AvaII restriction enzyme to generate the Δ327 construct or the FauI restriction enzyme to generate the Δ363 vector. The TTTCAAAA-mutant was generated by QuikChange Site-Directed Mutagenesis Kit (A13282, Thermofisher, La Jolla, CA, USA).
8
Plasmid Constructs for Drosha, Dicer, and eIF4H
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pCK-FLAG-TN Drosha and pCK-FLAG-TN Dicer were generous gifts from V. Narry Kim. pCK-FLAG-Drosha and pCK-FLAG-Dicer were previously generated in our lab [37] (link). FLAG-DGCR8 (Addgene) was purchased. The wild-type eIF4H minigene, comprised of exons 4–6 and the intervening introns, was generated by amplifying human genomic DNA with LongAmpTaq DNA polymerase (New England Biolabs). The amplicon was inserted into the pTarget vector (Promega) as per manufacturer's instructions. Mutations were introduced with the QuikChange Lightning mutagenesis kit (Agilent). Primer sequences are provided in Table S1 . All plasmid constructs were sequenced to verify the correct insertion sequence.
9
Construction of Δ1D2A-GLuc/SGLuc Chimeras
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Example 19
For the construction of Δ1D2A-GLuc/SGLuc chimeras, a nucleotide sequence encoding the Δ1D2A-SGLuc sequence was synthesized by Genescript in the pUC57 kan vector. PCR amplification was performed using OneTaq 2× Master Mix with Standard Buffer (New England Biolabs) and primers AscI-Kzk-2A-F (SEQ ID NO: 185) and Gluc-R-NotI (SEQ ID NO: 186) per manufacturer's instructions. Insertion into the pTarget vector (Promega) followed manufacturer's instructions for T/A cloning. Confirmation of insertion was performed by sequencing with primers T7 (SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180) and analysis of sequencing data was analyzed by Sequencher 4.8 program (Genecodes).
To construct the Δ1D2A-Gluc chimera the pTarget Δ1D2A-SGLuc construct was used as a template for site directed mutagenesis using the GENEART Site-Directed Mutagenesis System (Invitrogen) as per manufacturer's instructions with primers Gluc8990-MF-2 (SEQ ID NO: 197) and Gluc8990-MR-2 (SEQ ID NO: 198). Confirmation of mutation was performed by sequencing with primers T7 (SEQ ID NO: 179) and Seq-R (SEQ ID NO: 180) and analysis of sequencing data was analyzed by Sequencher 4.8 program (Genecodes).
10
Cloning and Expression of FYCO1 and Crystallins
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cDNAs for human FYCO1 and αB-crystallin were amplified by RT-PCR using human heart total RNA (Clontech), and subcloned into expression vectors. The identity of the PCR products was confirmed by sequencing analysis. Human αA-crystallin cDNA was kindly provided by Dr. N. Fujii (Kyoto University). FLAG-tagged human FYCO1, Myc-tagged human αA-crystallin, and Myc-tagged human αB-crystallin were subcloned into the pTARGET vector (Promega) for transient gene expression.
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