An inducible mammalian expression construct encoding NCAPH was obtained by cloning NCAPH into a pRetroX-Tight-Puro vector (Clontech #632104). The MCF-7 and BT-549 inducible NCAPH systems were generated by co-transfecting pRetroXTet-On Advanced with pRetroX-Tight-Puro-NCAPH. NCAPH expression was induced in cells after exposure to doxycycline (10 μg/ml).
Pretro x tight puro vector
Manufactured by Takara Bio
The PRetro-X-Tight(puro) vector is a lentiviral vector designed for the stable expression of genes of interest in mammalian cells. The vector contains a puromycin resistance gene, allowing for the selection of transduced cells. The vector's core function is to facilitate the introduction and integration of genetic material into the target cell's genome.
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Lab products found in correlation
5 protocols using pretro x tight puro vector
1
Inducible NCAPH Expression in Breast Cancer
2
Engineered Organelle-Specific Plasmids
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Site-directed mutagenesis of pLenti-CMV-Neo-PINK1 (C125G)-EYFP or pLVX-puro-OMA1 (E328Q)-EYFP was performed by PCR amplification (CloneAmp HiFi PCR Premix, Takara or Q5 High-Fidelity DNA Polymerase system, NEB) of PINK1 or OMA1 encoding plasmid using appropriate primers followed by Gibson assembly (In-Fusion HD Cloning system, Clontech) into the SalI-XhoI of the pLenti-CMV-Neo vector, or into the EcoRI site of pLVX-puro vector (Clontech). pLVX-puro-DELE1-HA and pLVX-puro-Su9-mCherry was created by PCR amplification and subcloning into the EcoRI site of pLVX-puro vector. pRetroX-Tight-puro-DELE1-HA was created by PCR amplification and subcloning into the BamHI/NotI site of pRetroX-Tight-puro vector (Clontech). All constructs were confirmed by DNA sequencing.
3
Cloning HA-tagged Dlg1 N-terminus
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The N-terminal region of Dlg1 was amplified by PCR, and an HA tag added to the sequence of the reverse primer, along with restriction sites for insertion into the pRetro-X-Tight(puro) vector (Clontech). Correct insertion was verified by Sanger sequencing. Primers were: Dlg66 forward, 5′-TTTTGCGGCCGCATGCCGGTCCGGAAGCAA-3′; Dlg66 reverse, 5′-ACGTCTTAAGTCAAGCGTAATCTGGAACATCGTATGGGTATGGTTCACACTGCTTTGAATGA-3′.
4
Cloning and Characterization of Dlg1 Constructs
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Full-length, HA-tagged WT-Dlg1 and the truncated Δ66-Dlg1 were amplified by PCR from a pCMV-SPORT6 vector (purchased from Open Biosystems) using the following primers: WT-Dlg1 forward, 5′-TTTTGCGGCCGCATGCCGGTCCGGAAGCAA-3′; Δ66-Dlg1 forward, 5′-TTTTGCGGCCGCATGTGTGTGGATCATTCAAAG-3′; Dlg1 reverse, 5′-AAAAAGAATTCTCAAGCGTAATCTGGAACATCGTATGGGTAAGCG-3′. Primers contained restriction sites for insertion into the pRetro-X-Tight(puro) vector (Clontech). Correct insertion was verified by Sanger sequencing.
5
Inducible NCAPH Expression System
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An inducible mammalian expression construct encoding NCAPH was obtained by cloning NCAPH into a pRetroX‐Tight‐Puro vector (Clontech #632104). The MCF‐7 and BT‐549 inducible NCAPH systems were generated by co‐transfecting pRetroXTet‐On Advanced with pRetroX‐Tight‐Puro‐NCAPH. NCAPH expression was induced in cells after exposure to doxycycline (10 μg/mL).
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