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N174 mcs

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The N174-MCS is a plasmid vector designed for cloning and expression of genes in bacterial systems. It contains a multiple cloning site (MCS) for insertion of DNA sequences, as well as elements necessary for plasmid replication and selection in E. coli.

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Lab products found in correlation

Plenti c mgfp, by OriGene (2 mentions) Lenticrisprv2 blast plasmid, by Addgene (2 mentions) Plenti cd1d1 mgfp, by OriGene (2 mentions) Plenti ifi204 myc ddk puro, by OriGene (2 mentions) Fastdigest protocol, by Thermo Fisher Scientific (2 mentions) Nebuilder hifi dna assembly cloning kit, by New England Biolabs (2 mentions) Geneticin g418, by InvivoGen (1 mentions)

3 protocols using n174 mcs

1

Rescue of lncRNA Knockdown

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The expression of selected lncRNAs was rescued by stable transduction of the mature lncRNA sequence of each gene into cells with confirmed KD of the gene. The lncRNA sequences (provided in Additional file 5: Table S4) were synthesized by Twist Bioscience (San Francisco, CA, USA) in their Twist Cloning Vector. The sequences were then ligated into N174-MCS (kindly provided by Adam Karpf; Addgene plasmid #81061). Lentiviral transduction was performed as described above into K562-CRISPRi cells with confirmed KD of the same lncRNA, followed by antibiotic selection with 600 μg/ml Geneticin (G418; Invivogen, Toulouse, France) for 7–10 days.
Hazan J.M., Amador R., Ali-Nasser T., Lahav T., Shotan S.R., Steinberg M., Cohen Z., Aran D., Meiri D., Assaraf Y.G., Guigó R, & Bester A.C. (2024). Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6’s role in hematopoietic differentiation and leukemia. Journal of Biomedical Science, 31, 27.
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2

Lentiviral CD1d1 and IFI204 constructs

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A lentiviral murine Cd1d1 expression construct (pLenti-Cd1d1-mGFP, Cat#MR226027L4) and its matched control construct (pLenti-C-mGFP, Cat#PS100093) were obtained from Origene. Murine Ifi204 expression vector (pLenti-Ifi204-Myc-DDK-Puro, Cat#MR222527L3), together with its control vector (pLenti-C-Myc-DDK-Puro, Cat#PS100092) were also purchased from Origene, and the puromycin selection cassette of these two Origene plasmids were replaced by blasticidin from lentiCRISPRv2-blast plasmid (Addgene#98293) using NEBuilder HiFi DNA Assembly Cloning kit (NEB, Cat#E5520S). For the LLC-1 experiment, the murine Cd1d1 was cloned into the receiving vector N174-MCS (Addgene#81061) with the restriction enzymes EcoR1 and Mlu1, using the FastDigest protocol of Thermo Scientific. All final construct sequences were confirmed by Sanger sequencing. Plasmids generated in this study are available upon written request.
Guo H., Vuille J.A., Wittner B.S., Lachtara E.M., Hou Y., Lin M., Zhao T., Raman A.T., Russell H.C., Reeves B.A., Pleskow H.M., Wu C.L., Gnirke A., Meissner A., Efstathiou J.A., Lee R.J., Toner M., Aryee M.J., Lawrence M.S., Miyamoto D.T., Maheswaran S, & Haber D.A. (2023). DNA hypomethylation silences antitumor immune genes in early prostate cancer and CTCs. Cell, 186(13), 2765-2782.e28.
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3

Lentiviral CD1d1 and IFI204 constructs

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A lentiviral murine Cd1d1 expression construct (pLenti-Cd1d1-mGFP, Cat#MR226027L4) and its matched control construct (pLenti-C-mGFP, Cat#PS100093) were obtained from Origene. Murine Ifi204 expression vector (pLenti-Ifi204-Myc-DDK-Puro, Cat#MR222527L3), together with its control vector (pLenti-C-Myc-DDK-Puro, Cat#PS100092) were also purchased from Origene, and the puromycin selection cassette of these two Origene plasmids were replaced by blasticidin from lentiCRISPRv2-blast plasmid (Addgene#98293) using NEBuilder HiFi DNA Assembly Cloning kit (NEB, Cat#E5520S). For the LLC-1 experiment, the murine Cd1d1 was cloned into the receiving vector N174-MCS (Addgene#81061) with the restriction enzymes EcoR1 and Mlu1, using the FastDigest protocol of Thermo Scientific. All final construct sequences were confirmed by Sanger sequencing. Plasmids generated in this study are available upon written request.
Guo H., Vuille J.A., Wittner B.S., Lachtara E.M., Hou Y., Lin M., Zhao T., Raman A.T., Russell H.C., Reeves B.A., Pleskow H.M., Wu C.L., Gnirke A., Meissner A., Efstathiou J.A., Lee R.J., Toner M., Aryee M.J., Lawrence M.S., Miyamoto D.T., Maheswaran S, & Haber D.A. (2023). DNA hypomethylation silences antitumor immune genes in early prostate cancer and CTCs. Cell, 186(13), 2765-2782.e28.
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