shRNA plasmids for UBR7 with pLKO.1-puro backbone (Sigma-Aldrich) were screened for efficient knockdown. Two of seven shRNAs were selected for subsequent experiments. Their sequences are as follows: UBR7-sh1 5′-CAGTGCACCCAGGGTTATTTG-3′ and UBR7-sh2–5′-GCTTAAAGCTAAGCAGCTTAT-3′. UBR7-sh1 targets the 3′-UTR of the gene, so the overexpression constructs were resistant to the shRNAs. HEK293T cells were plated at a density of 3 × 105 cells in 10-cm dishes. Eight micrograms of shRNA and packaging vectors were transfected into the cells as described previously47 (link). Transduced cells were selected using puromycin (10 μg/ml; Sigma) for 3 days.
Plko 1 puro backbone
Manufactured by Merck Group
The PLKO.1-puro backbone is a plasmid vector commonly used for lentiviral vector production. It contains the necessary genetic elements for packaging, transduction, and selection of cells expressing the gene of interest.
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Lab products found in correlation
2 protocols using plko 1 puro backbone
1
Efficient knockdown of UBR7 using shRNA
2
Targeted Gene Knockdown via shRNA Induction
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