A PCR product containing the sequence of tdTomato (vector ptdTomato; #63-2531, TaKaRa Clontech) was cloned into the lentiviral expression vector pLVX-IRES-neo (LentiX-Bicistronic Expression System; #63-2181, TaKaRa Clontech) to generate a pLVX-tdTomato-IRES-Neo construct. Notably, a resistance-sequence for G418-sulfate is contained in the lentiviral expression vector. The resulting nucleotide pLVX-tdTomato-IRES-Neo was verified by Sanger sequencing and restriction enzyme digestion.12 (link) LL/2 was transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). tdtLL/2 was enriched by cultivation in selection medium containing G418-sulfate (#A2912; Biochrom) and by repetitive FACS sorting. As previously described in detail,13 (link) tdtLL/2 cells were stably transduced with a pMXs vector containing the full-length murine EpCAM (UNIPROT entry: #Q99JW5) cDNA to generate the EpCAM-overexpressing cell line EpCAM/tdtLL/2.
Protocol detail
Generation of tdTomato and EpCAM-Expressing LL/2 Cells
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Cdna
Cell line
Cells
Digestion
Epcam
G418
Ires
Lipofectamine
Murine
Nucleotide
Restriction enzyme
Sulfate
Tdtomato
Vector
Corresponding Organization : Ludwig-Maximilians-Universität München
Other organizations : Center for Integrated Protein Science Munich, Walter and Eliza Hall Institute of Medical Research, Institute of Medical Microbiology and Hygiene
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Variable analysis
independent variables
- Transfection of LL/2 cells with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000)
- Enrichment of tdTomato-expressing LL/2 cells (tdtLL/2) by cultivation in selection medium containing G418-sulfate
- Stable transduction of tdtLL/2 cells with pMXs vector containing the full-length murine EpCAM cDNA to generate the EpCAM-overexpressing cell line EpCAMtdtLL/2
- Lentiviral expression vector pLVX-IRES-neo containing G418-sulfate resistance sequence
- Sanger sequencing and restriction enzyme digestion to verify the resulting nucleotide pLVX-tdTomato-IRES-Neo
- Lentiviral expression vector pLVX-IRES-neo containing G418-sulfate resistance sequence
- Not explicitly mentioned
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