R1 ESCs cultured in standard medium on gelatin coated p60 plates, were transfected with 3 μg pLKO.1-puro derived vectors (Sigma), expressing shRNA targeting Nanog (SHCLND-XM_132755), Oct4 (SHCLND-NM_013633) or eGFP (SHC005), which was used as control vector. Transfection was carried out using PEI, as transfection reagent, with a DNA/PEI ratio of 1:5. After ON incubation, the medium was replaced by fresh medium. Twenty four hours after transfection, transfected cells were selected for 48 h with puromycin at 3 μg/ml final concentration. Then, total RNA was isolated using TRIzol reagent (Life Technologies) and mRNA expression was analyzed by RT-qPCR as described in the RT-qPCR section.
Plko 1 puro derived vectors
Manufactured by Merck Group
PLKO.1-puro derived vectors are plasmid-based tools used for gene expression and knockdown applications in cell lines. These vectors contain a puromycin resistance gene, allowing for the selection of cells that have successfully integrated the vector. The core function of these vectors is to facilitate genetic manipulation and screening experiments in cultured cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using plko 1 puro derived vectors
1
Knockdown of Pluripotency Factors in mESCs
2
Knocking down Nanog and Oct4 in ESCs
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R1 ESCs cultured in standard medium on gelatin coated p60 dishes were transfected with 3 μg pLKO.1-puro derived vectors (Sigma), expressing shRNA targeting Nanog (SHCLND-XM_132755), Oct4 (SHCLND-NM_013633) or eGFP (SHC005), which was used as control vector. Transfection, selection and mRNA expression analysis were carried out as previously described [48] (link).
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