Mouse Tulp1, Tulp2, Tulp3, and TUB cDNAs were cloned into the pIRES2-DsRed2 vector (Clontech plasmid #632420), which expresses both the gene of interest and the dsRed gene. TUB PIP2- and IFT-A-mutant cDNAs were cloned into the pLVX-EF1α-IRES-ZsGreen vector (Clontech plasmid #631982), which expresses both the gene of interest and the ZsGreen gene. To examine the subcellular localization of TULPs in RPE1 cells, mouse Tulp1, Tulp2, Tulp3, TUB, and Tulp1IFT-A(+) cDNAs were cloned into the pCMV-Tag3B vector (Agilent Technologies plasmid #211173), which makes it possible to visualize the expression of a gene of interest using an anti-Myc antibody.
Plvx ef1α ires zsgreen vector
Manufactured by Takara Bio
The PLVX-EF1α-IRES-ZsGreen vector is a lentiviral expression vector that drives the expression of the ZsGreen fluorescent protein under the control of the EF1α promoter. The internal ribosome entry site (IRES) allows for the expression of the ZsGreen gene as a separate transcript from the gene of interest.
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3 protocols using plvx ef1α ires zsgreen vector
1
Subcellular Localization of Tulp Proteins
2
Generating NPM1 Mutant Lentivector
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To construct NPM1 lentivector the mutant NPM1 cDNA (mutation type A, TCTG duplication) was cloned into the pLVX-EF1α-IRES-ZsGreen vector (Clontech) using EcoRI and BamHI restriction sites.
A third-generation packaging system was used to produce viral particles. Lentiviral supernantants for NPM1-expressing virus and control virus (empty pLVX-EF1α-IRES-ZsGreen vector) were produced in 293T cells by Ca3PO4 cotrasfection of the plasmids as previously described (De Palma and Naldini, 2002 (link)). C1498 cell line was infected using viral supernatants at 1:2 ratio with RPMI complete medium. Percentage of infection was evaluated by flow cytometry for GFP expression. Subsequently, infected C1498 cells were sorted by FACSAria to obtain pure populations (100% GFP expression).
A third-generation packaging system was used to produce viral particles. Lentiviral supernantants for NPM1-expressing virus and control virus (empty pLVX-EF1α-IRES-ZsGreen vector) were produced in 293T cells by Ca3PO4 cotrasfection of the plasmids as previously described (De Palma and Naldini, 2002 (link)). C1498 cell line was infected using viral supernatants at 1:2 ratio with RPMI complete medium. Percentage of infection was evaluated by flow cytometry for GFP expression. Subsequently, infected C1498 cells were sorted by FACSAria to obtain pure populations (100% GFP expression).
3
Generating NPM1 Mutant Lentivector
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To construct NPM1 lentivector the mutant NPM1 cDNA (mutation type A, TCTG duplication) was cloned into the pLVX-EF1α-IRES-ZsGreen vector (Clontech) using EcoRI and BamHI restriction sites. A third-generation packaging system was used to produce viral particles. Lentiviral supernantants for NPM1expressing virus and control virus (empty pLVX-EF1α-IRES-ZsGreen vector) were produced in 293T cells by Ca3PO4 co-trasfection of the plasmids as previously described (26) . C1498 cell line was infected using viral supernatants at 1:2 ratio with RPMI complete medium. Percentage of infection was evaluated by flow cytometry for GFP expression. Subsequently, infected C1498 cells were sorted by FACSAria to obtain pure populations (100% GFP expression).
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