To stably knockout IQGAP1 expression in MDA-MB-231, CRISPR-Cas9 genome editing method was used44 (link). Two guide RNA sequences, 5′-CCCGTCAACCTCGTCTGCGG-3′ and 5′-GGCGTGGCCCGGCCGCACTA-3′, that target the first exon of human IQGAP1 gene were cloned into PX459V2.0 vector (Addgene, Watertown, MA, USA). Constructs were transfected for 36 h and then transiently selected with 1 μg/ml puromycin (Millipore Sigma). After 48 h incubation, puromycin was removed and single cells were seeded in 96-well tissue culture dishes. Cells were expanded and positive colonies were selected by immunoblotting with specific antibodies against human IQGAP1. Successful editing of the IQGAP1 gene was further validated by genomic sequencing. Multiple clones of IQGAP1 knockout cells were tested to avoid clonal variation.
Px459v2.0 vector
Manufactured by Addgene
Sourced in United States
PX459V2.0 is a plasmid vector used in molecular biology research. It serves as a backbone for CRISPR-Cas9 gene editing experiments. The vector contains the Cas9 coding sequence and a cloning site for guide RNA insertion. PX459V2.0 allows for targeted genetic modifications in cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (1 mentions)
Site directed mutagenesis, by New England Biolabs (1 mentions)
Gateway technology, by Thermo Fisher Scientific (1 mentions)
Puromycin, by InvivoGen (1 mentions)
4 protocols using px459v2.0 vector
1
Stable IQGAP1 Knockout in MDA-MB-231 Cells
2
Lentiviral Overexpression and CRISPR Editing
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The entry vector pDONR223 containing the full-length GRN (1–1179 base pairs) from the human orfeome collection was used to engineer a stop codon by site-directed mutagenesis (New England Biolabs) at the end of the open-reading frame sequence (ORF). Gateway technology (Thermo Fisher) was used to transfer the GRN ORF with LR cloning from the entry vector to the pHAGE lentiviral destination expression vector. sgRNA sequences for editing the TMEM192, GRN, HEXA, NPC1 and NPC2 loci were cloned into the pX459 V2.0 vector (Addgene Cat#62988) as described31 (link).
3
Knockdown of mTORC Components via CRISPR
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Knockdowns were done using the PX459 V2.0 vector (Addgene) containing sgRNAs targeting CCTε, Raptor or PhLP1. The same vector with a nontargeting sgRNA segment was used as a negative control. The sgRNA sequences are provided in Supplementary Table 4 . These vectors were transfected into HEK-293T cells at 25–40% confluency with Lipofectamine 3000 (Thermo Fisher Scientific) and then treated with 1 μg/mL puromycin (Invivogen). Forty-eight hour after transfection of PX459V2.0, cells were transfected with vectors containing mTORC or Gβγ components. Cells were harvested for immunoprecipitation and immunoblotting 96 h after addition of the sgRNA vector.
4
CRISPR-Cas9 Mediated Ext1 Knockout
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The CRISPR–Cas9 system was introduced to generate Ext1-heterozygous deleted 3T3-L1 (Ext1+/−) cells. The oligonucleotides corresponding to Ext1 exon 1, containing the protospacer adjacent motif sequence, were inserted into the BbsI restriction site of the pX459 v2.0 vector (plasmid number #62988; Addgene). The guide RNA sequences used in this study were designated using CRISPR direct (48 (link)) and are shown in Table S1 . Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, 2.5 × 105 cells per well were seeded in a 6-well plate before transfection and incubated overnight with 250 μl of a mixture of Lipofectamine 2000 and Opti-MEM. After that, we selected the puromycin-resistant stable transformants for further culture. After the dideoxy sequencing of isolated genomic DNA from the transformants to confirm the genome editing, Ext1+/− cells were established.
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