A construct overexpressing FGF14-AS2 was generated by ligating the full-length human FGF14-AS2 into vector pLVX-EF1α-IRES-puro (Clontech, Mountain View, CA, USA). For FGF14-AS2 knockdown, shRNA oligos against FGF14-AS2 were synthesized, annealed, and ligated into vector pLentilox 3.7 (Addgene, Cambridge, MA, USA), with a nontargeting control sequence (shNC) as the control. Table S3 lists the primers used. High-titer lentivirus was packaged in HEK 293T cells. The viral particles were collected by centrifugation at 48 h post transfection, and applied to cells in the presence of 5 μg/mL polybrene for 48 h. FGF14-AS2-overexpressing cells were selected using puromycin (5 μg/mL) for 2 weeks. Gene knockdown and overexpression were confirmed by qRT-PCR.
Plentilox3
Manufactured by Addgene
Sourced in United States
PLentiLox3.7 is a lentiviral vector designed for gene delivery and expression. It contains a cytomegalovirus (CMV) promoter, a multiple cloning site for inserting genes of interest, and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to enhance transgene expression.
Automatically generated - may contain errors
Lab products found in correlation
Plentilox3.7 ef1α mcherry vector, by Addgene (2 mentions)
Plvx ef1α ires puro, by Takara Bio (1 mentions)
Peiz hiv zsgreen lentivirus vector, by Addgene (1 mentions)
Retronectin, by Takara Bio (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Polyethyleneimine, by Polysciences (1 mentions)
Lentivirus vectors, by System Biosciences (1 mentions)
4 protocols using plentilox3
1
Overexpression and Knockdown of FGF14-AS2
2
Lentiviral Constructs for miR-221 and QKI-5
3
Lentiviral Transduction of Pluripotent Stem Cells
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We utilized the lentiviral vector pLentiLox 3.7 (pLL3.7, Addgene plasmid 11795) (23 (link)) expressing the mouse scrambled shRNA or shGata6 (sequence HpaI-sh Gata6 -5p-F TG-CGGTC TCTACAGCAAGATG-TTCAAGAGA-CATCTTGCTGTAGAGA CCG-CTTTTTTC, XhoI-sh Gata6 -5p-R TCGAGAAAAAAG-CGG TCTCTACAGCAAGATG-TCTCTTGAA-CATCTTGCTGTAGAG ACCG-CA) (Sigma Aldrich) which has GFP as a selection marker. For the generation of lentivirus, we transfected HEK293T cell with 20 μg of a lentiviral vector, 10 μg of PLP1 (Invitrogen), 10 μg of PLP2 (Invitrogen), 10 μg of VSVG (Invitrogen) using a transfection agent, polyethyleneimine (Polysciences). After 24 hours, we changed the medium with DMEM/high glucose (Gibco) supplemented with 10% FBS (Gibco). After 24 hours, we harvested the supernatant and added 10 ml of growth medium for the second harvest. For lentivirus transduction, we prepared retronectin (TaKaRa, Japan)-coated 35 mm dish. We seeded 2 × 105 PE-iPSC or mESC on the dish with 1.5 ml virus supernatant and 1.5 ml fresh growth medium. After 24 hours, we collected the whole cells in the dish and re-seeded on a dish with STO feeder layer. After 7 days, we mechanically selected undifferentiated colonies expressing GFP. We dissociated the colonies with 0.25% Trypsin/EDTA (Gibco) and then seeded them on a dish with STO feeder layer.
4
Lentiviral Overexpression of Anti-miR-93 in Breast Cancer Cells
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The sequence of precursor miR‐93 (mature miR‐93 and its 5′‐ and 3′‐ flanking regions) and the full‐length coding region of the WASF3 mRNA (NM_006646.6 [GenBank]) were amplified by PCR and cloned into the pEIZ‐HIV‐ZsGreen or mCherry lentivirus vector (Addgene: #18121) or the pLentiLox3.7‐EF1α‐mCherry vector, a derivative of pLentiLox3.7 (Addgene: #11795), respectively.18 The lentivirus vectors encoding for the anti‐miR‐93 construct (miRZip‐93) and a nontargeting control (negative control) were purchased from System Biosciences. Lentiviruses were produced as previously described.24 Breast cancer cells were infected with lentivirus constructs at a MOI of 5.
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