Previously reported shRNA target sequences for Ets1 and Runx1 (36 (link), 37 (link)) were cloned into the pSIREN shuttle vector (Clontech), excised as BglII-MluI fragments, and inserted into pcDNA3.1 containing a truncated human CD4 cDNA (38 (link)). P5424 cells were cultured as described (39 (link)) and were transfected by electroporation (Biorad, 250 V/960 μF ) with control (scrambled GFP 5′-AAGCTGGAGTACAACTACA-3′), ETS1-specific (target sequence 5′-GCAGACAGACTACTTTGCCAT-3′), or RUNX1-specific shRNA vectors (target sequence 5′-GCCCTCCTACCATCTATACTA-3′). Transfected cells were purified 48 hours post-transfection with an EasySep Human CD4+ T Cell Enrichment Kit (Stem Cell Technologies #19052).
Protocol detail
Silencing Ets1 and Runx1 in P5424 Cells
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Cd4 t cell
Cdna
Cells
Electroporation
Human
Runx1
Shrna
Shuttle vector
Stem cell
Transfection
Vectors
Corresponding Organization : Washington University in St. Louis
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Variable analysis
independent variables
- Transfection with control (scrambled GFP 5′-AAGCTGGAGTACAACTACA-3′) shRNA vector
- Transfection with ETS1-specific (target sequence 5′-GCAGACAGACTACTTTGCCAT-3′) shRNA vector
- Transfection with RUNX1-specific (target sequence 5′-GCCCTCCTACCATCTATACTA-3′) shRNA vector
- Protein or gene expression levels of Ets1 and Runx1
- P5424 cell line cultured as described in reference 39
- Electroporation conditions (250 V/960 μF)
- Time point (48 hours post-transfection) for purification of transfected cells
- EasySep Human CD4+ T Cell Enrichment Kit (Stem Cell Technologies #19052) used for purification of transfected cells
- Positive control: Transfection with control (scrambled GFP 5′-AAGCTGGAGTACAACTACA-3′) shRNA vector
- Negative controls: Not explicitly mentioned
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