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Pbabe puro control

Manufactured by Addgene

The PBabe-puro-Control is a plasmid vector that can be used as a control in various cell-based experiments. It contains a puromycin resistance gene, which allows for the selection of cells that have successfully integrated the vector.

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2 protocols using pbabe puro control

1

Knockdown of S100A10 Using shRNA

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The S100A10 shRNA1 knockdown construct was designed by cloning the following dsRNA oligo 5′-GAT CCC CGT GGG CTT CCA GAG CTT CTT TCA AGA GAA GAA GCT CTG GAA GCC CAC TTT TTA-3′ and 5′-AGC TTA AAA AGT GGG CTT CCA GAG CTT CTT CTC TTG AAA GAA GCT CTG GAA GCC CAC GGG-3′ into the pSUPER-retro-puro vector plasmid (OligoEngine). The non-silencing siRNA (4390843) and S100A10 siRNA (s12429) were purchased from the Ambion Silencer Select pre-designed and validated siRNA library (ThermoFisher Scientific). The plasmid vectors pBabe-puro-Control (#1764) and pBabe-puro-FOXC2 (#15535) were obtained from the plasmid depository Addgene. The pGIPZ SMAD4 and FOXC2 constructs were obtained from EGAD (enhanced Gene Analysis and Discovery) core facility at Dalhousie University. All transfected cell lines were selected and maintained in 1 µg/ml puromycin.
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2

Retroviral and Lentiviral Transduction of hUCB-MSCs

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The retroviral plasmid vectors, including pBABE-puro–control, pBABE-puro–GFP–wt-LMNA, pBABE-puro–GFP–progerin, shCTL-mLPx and mLPx-shZMPSTE24, were purchased from Addgene. The pInducer20-GATA4 and MSCV-GATA4 shRNA were provided by Dr. Stephen J. Elledge of Harvard medical school24 (link). Virus production and transduction were performed as previously described30 (link). Briefly, the retroviral plasmids pBABE-puro–control, pBABE-puro–GFP–wt-LMNA, pBABE-puro–GFP–progerin, shCTL-mLPx, mLPx-shZMPSTE24, and MSCV-GATA4 shRNA were transfected into 293FT cells with VSV-G and gag/pol plasmids using Fugene 6 transfection reagent (Roche, Basel, Switzerland). The viral supernatants were collected 48 and 72 h after transfection and were used to infect hUCB-MSCs in the presence of 5 μg/ml polybrene (Sigma). After the viral transduction, the hMSCs were selected with puromycin (0.5 μg/ml) for 4 days and cultured for 3 days before the analysis. The lentiviruses were generated according to the manufacturer (Thermo Fisher scientific, MA, USA) to introduce Dox-inducible flag-GATA4. The viral supernatants were collected 48 and 72 h after transfection and were used to infect hUCB-MSCs (MOI = 2) in the presence of 5 μg/ml polybrene (Sigma). At 24 h after transduction, the cells were washed four times in PBS and maintained in growth medium.
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