The GALNT3 KD and control A2780s clones used in this study were previously generated, as described [14 (link)]. Briefly, a shRNA targeting the GALNT3 sequence 5′-TACTGCTGAAGGAAATCAT-3′ was designed using the siRNA Ambion Target Finder software (http://www.ambion.com/techlib/misc/siRNA_finder.html ), and subcloned into the pSilencer 4.1 puro vector (Ambion). A2780s cells were stably transfected with this construct and clones were isolated as previously shown [14 (link)]. In addition, A2780s cells were mock-transfected with the pSilencer 4.1 puro vector, and the stably-transfected clones were isolated and used as controls.
Psilencer 4.1 puro vector
Manufactured by Thermo Fisher Scientific
The PSilencer 4.1 puro vector is a plasmid designed for gene silencing and knockdown experiments. It contains a puromycin resistance gene for selection of successfully transfected cells. The core function of this vector is to facilitate the expression of short hairpin RNA (shRNA) sequences, which can target and silence specific genes of interest.
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Lab products found in correlation
2 protocols using psilencer 4.1 puro vector
1
Generation and Characterization of GALNT3 Knockdown Cells
2
Stable Knockdown of GALNT3 in A2780s Cells
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A shRNA, targeting the GALNT3 sequence 5'-TACTGCTGAAGGAAATCAT-3', was designed using the siRNA Ambion Target Finder software (http://www.ambion.com/techlib/misc/siRNA_finder.html ), and subcloned into the pSilencer 4.1 puro vector (Ambion). A2780s cells were stably transfected with the shRNA-GALNT3 plasmid using the ExGen 500 transfection reagent (Fermentas Canada Inc., Burlington ON), according to the manufacturer's instructions. Cells were consecutively grown for 2 weeks in selection medium containing 5 μg/ml puromycin (Wisent, Canada) to isolate stable clones. Cells were also mock-transfected with the pSilencer 4.1 puro vector, and the stably-transfected clones were isolated as controls. Stable clones with inhibited GALNT3 expression were evaluated and validated by semi-quantitative RT-PCR and Western blot.
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