GIPZ lentiviral shRNA particles were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). KU812 cells (90,000) were transduced with 20 MOI of lentivirus particles in serum-free media for 4 hr and then 10% fetal bovine serum was added. Puromycin (0.6 µg/ml) was added on day 2 and selection performed for 5 days. The cells were harvested for RT-qPCR using gene-specific primers purchased from SuperArray (Qiagen, Valencia, CA); relative gene expression levels were calculated using the 2−ΔΔCT method. After lentiviral transduction fluorescent activated cell sorting (FACS) was performed at 48 hr to determine transfection efficiency. The percentage of green fluorescence protein (GFP) positive cells was used to normalize the qPCR data.
Gipz lentiviral shrna particles
Manufactured by Thermo Fisher Scientific
GIPZ lentiviral shRNA particles are a laboratory tool designed for gene silencing experiments. They contain short hairpin RNA (shRNA) sequences that can be used to knockdown the expression of specific target genes in cell lines and primary cells. The particles can be used to transduce cells and stably express the shRNA, enabling researchers to study the effects of gene knockdown.
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2 protocols using gipz lentiviral shrna particles
1
Lentiviral shRNA Knockdown Assay
2
Lentiviral shRNA Targeting mANO1 in mpkCCD Cells
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GIPZ lentiviral shRNA particles were obtained from Thermo Fisher Scientific and targeted to mANO1 (V2LMM_92063), GAPDH (RHS4372), or negative nonsilencing control (RHS4348). For lentiviral transduction, mpkCCD14 cells were plated at 8 × 105 cells/plate in 60-mm dishes and allowed to grow overnight. After 24 h, cells were switched to serum-free medium with lentiviral particles. At 24 h after transduction, infected cells were selected by the addition of 1.5 μg/ml puromycin in serum-containing medium. At 7 d after selection, cells were plated at 5 × 105 cells/filter in serum-containing medium. After 48 h, cells were switched to serum-free medium for an additional 48 h before fixation. Only cells identified as positively transduced by expression of TurboGFP were counted in the analysis of the percentage of ciliated cells and measurements of cilia lengths.
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