HEK293 cells were cultured in DMEM (Gibco, Life Technologies) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2. 24 h prior to transfection, cells were seeded at with 10 ml 2.5·105 cell/ml in 10 cM plates with tissue culture treated glass slides. Cells were transfected using FuGENE HD (Promega, Madison, WI, USA) transfection reagent following manufacturer’s protocol and 10 ng of plasmid DNA. Full length PRDM9 was cloned from mouse testis cDNA library into the pCEP4 expression vector (Invitrogen). A 3X-FLAG tag was inserted in frame replacing the 5′ start methionine using yeast based homologous recombination technology to allow detection of expressed protein.
Pcep4 expression vector
Manufactured by Thermo Fisher Scientific
The PCEP4 expression vector is a plasmid designed for the expression of recombinant proteins in Escherichia coli. It contains a strong promoter and a multiple cloning site, allowing for the insertion of a gene of interest and its subsequent expression in the bacterial host.
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Lab products found in correlation
4 protocols using pcep4 expression vector
1
PRDM9 Overexpression in HEK293 Cells
2
PRDM9 Allele Generation and Tagging
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The PRDM9B allele was purchased from OriGene (Rockville, MD). Oligonucleotide primers were designed to include a 5’ V5 epitope tag and used to amplify the full-length PRDM9 and cloned into pCEP4 expression vector (Invitrogen) to create pCB09. A 6X-HIS-3X-FLAG tag was inserted in frame replacing the V5-tag using yeast-based homologous recombination [53 (link)]. The zinc-finger arrays for both PRDM9A and PRDM9C were amplified from human genomic DNA [7 (link)] and cloned into pBAD-HisC (Invitrogen). These zinc-finger arrays were subcloned into the pCEP4 vectors using restriction enzymes AflII and HindIII (NEB) to create full-length tagged versions of FLAG-PRDM9C (pCB51), V5-PRDM9C (pCB47), and FLAG-PRDM9A (pCB53), and V5-PRDM9A (pCB48) for expression in mammalian cell culture. The V5-PRDM9C-G278A allele was created using QuikChange II site-directed mutagenesis (Agilent Technologies) to change glycine 278 to alanine to create pCB56. All cloning oligonucleotides are listed in S3 Table .
3
Stable cell line generation for DDR1
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Recombinant DDR1 constructs were cloned into pCEP4 expression vector (Invitrogen, Life Technologies). HEK293-EBNA cells were transfected with the pCEP4 vectors using TransIT-2020, and transfectants were selected by treatment with 800 μg/ml hygromycin B (Sigma-Aldrich) over 3 wk. A431 cells were also used to generate stable cell lines and selected with 400 μg/ml hygromycin B. Transfected stable cell lines were maintained in DMEM containing 5% FBS, penicillin/streptomycin, and hygromycin B (400 μg/ml for A431, 800 μg/ml for HEK293) at 37°C, 5% CO2.
4
Purification and Characterization of Recombinant TNC
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Recombinant his-tagged human TNC was purified as described (36, 37) . and used for incubation with cells. Murine strep-tagged TNC was used in negative EM microscopy and treatment of cells. For cloning murine TNC, a PCEP4 expression vector (Invitrogen, catalog number V04450) with TNC (NP_035737.2, aa: 174-2019) from Mus musculus has been obtained from R. Chiquet-Ehrismann (FMI, Basel, Switzerland). The coding sequence was modified with a BM40 signal peptide and a N terminal double strep II tag and was confirmed by sequencing (Supplemental Table S7). In order to generate stable cell lines, HEK293 EBNA cells were transfected with the expression vector using Fugene HD (Promega, catalog number E2311). After 48 hours of transfection, the medium was replaced with 0.5 µg/ml containing DMEM/F12 medium with 10% FCS and the cells were grown to confluency. The protein was then purified from the supernatant by using the Streptactin matrix (IBA, Lifesciences, catalog number 2-1021-001) following the manufacturer's guidelines and was then dialyzed 3 times against PBS as previously described (37) .
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