The THOR expression construct were generated by amplifying the full-length transcript from NCI-H1299 cells and subcloning into the pLenti6 expression vector (Invitrogen), LacZ constructs were used as controls. Following Sanger sequencing (University of Michigan Sequencing Core) confirmation of the inserts, lentiviruses were generated at the University of Michigan Vector Core. NCI-H1437 and SK-MEL-5 cells were infected with lentiviruses expressing THOR or LacZ and stable pools and clones were generated by blasticidin selection (Invitrogen). The THOR deletion constructs were also generated by amplifying by PCR using the full-length transcript as a template and were subcloned into the pLenti6 expression vector (Invitrogen).
Plenti6 expression vector
Manufactured by Thermo Fisher Scientific
The PLenti6 expression vector is a lentiviral vector designed for the stable integration and expression of genes of interest in target cells. The vector allows for the production of lentiviral particles, which can then be used to transduce and express the gene of interest in a wide range of cell types, including dividing and non-dividing cells. The core function of the PLenti6 expression vector is to facilitate the delivery and sustained expression of genetic material in transduced cells.
Automatically generated - may contain errors
Lab products found in correlation
Blasticidin selection, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Plko 1 shitga7, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using plenti6 expression vector
1
THOR Expression Construct Generation
2
Stable Transfection of CCL2 and CCR2
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Either full-length of CCL2 cDNA or CCR2 cDNA were amplified by polymerase chain reaction (PCR) and cloned into plenti6 expression vector (Invitrogen). Lipofectamine 2000 (Invitrogen) was used to stably transfect CCL2 and CCR2 into S26 and SUNE-1 cells, respectively. Blank vector-transfected cells were used as controls.
3
ITGA7 Overexpression and Knockdown Plasmid Construction
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ITGA7 overexpression plasmid was kindly provided by Dr. Jianhua Luo (Department of Pathology, University of Pittsburgh School of Medicine). Briefly, the full-length wild-type ITGA7 cDNA (NM_002206.2) was cloned into the pLenti6 expression vector (Invitrogen) and transfected into EC109 and KYSE30 cells with Lipofectamine 2000 Reagent (Invitrogen). Stable ITGA7-expressing clones were selected for 2 weeks with blasticidin (Sigma-Aldrich). EV-transfected cells were used as controls. For ITGA7 knockdown, the scrambled shRNA plasmid (pLKO.1-NTC) and the ITGA7-specfic shRNA expression vectors (pLKO.1-shITGA7) were purchased from Sigma-Aldrich. Two constructs against ITGA7 were used: shITGA7-1 (TRCN0000057708) and shITGA7-3 (TRCN0000057711). The shRNA sequences listed in Supplementary Table 7 . The pLKO.1-shITGA7 or the scrambled shRNA plasmid was transfected into KYSE180 and KYSE520, and stable clones were selected using puromycin (Sigma-Aldrich).
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