The coding sequence of human WASF2 was cloned into the expression vector pCDH‐CMV‐MCS‐EF1‐Puro (System Biosciences, Mountain View, CA, USA). The shRNAs against WASF2 were synthesized by Ribobio (Guangzhou, China) and inserted into the pLKO.1‐TRC cloning vector (Invitrogen). All constructs were verified by sequencing. A mixture of pCDH‐WASF2 or pCDH‐CMV‐MCS‐EF1‐Puro, pLKO.1‐shWASF2, or pLKO.1‐TRC cloning vector, and the adjuvant vectors psPAX2 and pMDG2, were transfected into HEK293T cells with the assistance of Lipofectamine 2000 reagent (Invitrogen) to generate lentiviruses. HGC‐27 and MKN‐45 cells were infected with the lentiviral particles according to the manufacturer's protocol. The empty vectors were packaged as negative controls. Stable transfectants were selected and cultured in medium containing 3 μg/mL puromycin.
Plko 1 trc cloning vector
Manufactured by Thermo Fisher Scientific
Sourced in China
The PLKO.1-TRC cloning vector is a lentiviral expression vector designed for the generation of stable cell lines through lentiviral transduction. It is a key tool for functional genomics studies, allowing the expression or knockdown of genes of interest. The vector contains the necessary elements for lentiviral packaging, transduction, and selection of transduced cells.
Automatically generated - may contain errors
2 protocols using plko 1 trc cloning vector
1
Stable Overexpression and Knockdown of WASF2
2
Stable Knockdown of hnRNPA1, CAV1, and PTBP1 in A549 Cells
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A549 cell line with stable knockdown of hnRNPA1, CAV1, or PTBP1 was established using pLKO.1-TRC Cloning Vector (Invitrogen) per the manufacturer’s protocol. Briefly, the short hairpin RNA (shRNA) primers targeting corresponding genes and control (CTR) oligo were respectively subcloned into lentiviral expression plasmid pLKO.1 between AgeI and EcoRI sites. The constructed plasmids were confirmed by DNA sequencing (Sangon Biotech) before use. Lentivirus supernatants were generated from HEK293T packaging cells. Puromycin was applied to screen stable cell lines. The sequences of shRNA primers are listed as follows (He et al., 2007 (link); Han et al., 2015 (link); Liu et al., 2016 (link)):
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