A LINC00472 transcript (2933 bp, NR_026807.1) was assembled and inserted in a lentiviral vector, pCDH-EF1-MCS-pA-PGK-copGFP-T2A-Puro (pCDH), as previously described (13 (link)). The sequence of the insert has been confirmed by sequencing. MB231 and Hs578T cells were transfected with the LINC00472 plasmid or an empty plasmid (pCDH vector only) using the Lipofectamine 3000 reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Cells with stable expression of LINC00472 were selected through puromycin screening (Thermo Fisher Scientific). To maintain stably transfected cells, puromycin was added into culture medium, and the puromycin-containing culture medium was replaced every 3 days. A single cell clone was also generated from the stable cell pool through the limiting dilution cloning. Plasmids (pCMV-ESR1) with and without the full-length of human ESR1 (NM_000125, #RC213277) and (pCMV-vector, #PS100001), respectively, were purchased from Origene Technologies, and the plasmids were transfected into the 293T cells and breast cancer cell lines using the Lipofectamine 3000 reagent (Themo Fisher Scientific).
Protocol detail
Overexpression of LINC00472 in Breast Cancer
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293t cells
Breast cancer cell lines
Cell
Culture medium
Dilution
Human
Lipofectamine
Plasmids
Puromycin
Vector
Corresponding Organization :
Other organizations : University of Hawaiʻi at Mānoa, University of Hawaii System, Tongren Hospital, Shanghai Jiao Tong University
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Variable analysis
independent variables
- Transfection of LINC00472 plasmid or empty plasmid (pCDH vector only) into MB231 and Hs578T cells
- Transfection of pCMV-ESR1 plasmid with or without full-length human ESR1 into 293T cells and breast cancer cell lines
- Stable expression of LINC00472
- Overexpression of ESR1
- Lipofectamine 3000 reagent for transfection
- Puromycin selection and maintenance of stably transfected cells
- Limiting dilution cloning to generate single cell clones
- Positive control: pCMV-ESR1 plasmid with full-length human ESR1
- Negative control: pCMV-vector (empty plasmid)
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