Stable A549 DANCR KD cells (3 × 105 cells/well of a 6-well plate) were cotransfected with phage-ubc-nls-ha-tdMCP-GFP (Addgene 40649) and either pcDNA3-MS2-DANCR or pcDNA3-MS2-Vector constructs using FuGeneHD according to manufacturer’s instructions. After 48 h of transfection, cells were lysed and incubated while rocking with anti-GFP conjugated beads for 2 h at room temperature. Pulldown products were released using Trizol, and RNA was extracted and used to measure miR-216a expression via qRT-PCR.
Phage ubc nls ha tdmcp gfp
Manufactured by Addgene
Phage-ubc-nls-ha-tdMCP-GFP is a plasmid construct that expresses a fusion protein containing the MS2 coat protein (MCP) tagged with a nuclear localization signal (NLS), hemagglutinin (HA) tag, and green fluorescent protein (GFP). This construct is driven by the ubiquitin (Ubc) promoter.
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3 protocols using phage ubc nls ha tdmcp gfp
1
DANCR Regulation via MS2-Tagged qRT-PCR
2
Modular PiggyBac Genetic Constructs
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The NLS SV40 -dCas9-(NLS SV40 ) X3 -(GCN4 -V4 ) X24 -NLS SV40 -P2A-BFP fragment was amplified by PCR from plasmid pHRdSV40-NLS-dCas9-(GCN4 -V4 ) X24 -NLS-P2A-BFP-dWPRE (Addgene Plasmid #60910) and then ligated into PiggyBac plasmid pB-TRE3G-BsmBI-EF1α-PuroR-P2A-rtTA (home-made) by Golden Gate assembly with BsmBI and T4 ligase (NEB). The scFV-sfGFP-GB1-NLS SV40 fragment was amplified from plasmid pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE (Addgene Plasmid #60906) and then ligated into PiggyBac plasmid pB-TRE3G-BsmBI-EF1α-HygroR-P2A-rtTA (home-made) by Golden Gate assembly with BsmBI and T4 ligase (NEB). The tdMCP fragment was amplified from phage-ubcnls-ha-tdMCP-gfp (Addgene Plasmid #40649), and the KRAB was amplified from pHAGE-EF1α-dCas9-KRAB (Addgene Plasmid #40649) and then ligated into plasmid pB-CMV (home-made). The tdPCP fragment was amplified from phageubc-nls-ha-tdPCP-gfp (Addgene Plasmid #40650), and the VPR was amplified from SP-dCas9-VPR (Addgene Plasmid #63798) and then ligated into plasmid pB-CMV (home-made). The telomere, rDNA10, MUC4-E2 targeting sgRNAs and other sgRNA expression plasmids were made according to procedures described previously (6) . All primers used in this work were listed in Supplementary Tables 12345678.
3
Cloning Mouse CDS and 3'UTR with CFP
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All mouse CDS and 3'UTR used were amplified from mouse cDNA using the primers indicated in Table 2. Fragments were cloned into phage-cmv-cfp-24xms2 vector (Addgene plasmid # 40651) created by Robert Singer lab. CDS fragments (from start codon to penultimate codon) were inserted in frame, upstream to CFP at the AgeI and NotI sites (under CMV promoter). 3'UTR fragments were inserted downstream to the MS2 stem loops cassette at the ClaI site. Constructs designated as 'Full' contain both the CDS and 3'UTR of the respective gene. 'CDS' or '3'UTR' designations indicate constructs with only these regions of the gene (cloned at the above restriction sites) and the CFP reporter. Proper construction was confirmed by sequencing and proper transcripts' length was validated by northern blot. We note that all constructs contained 12 MS loops instead of the expected 24, presumably due to recombination during cloning.
MCP-YFP plasmid was a gift from Prof. Yaron Shav-Tal (Bar Ilan University). MCP plasmid used for axonal live-imaging was phage-ubc-nls-ha-tdMCP-gfp (Addgene plasmid # 40649).
MCP-YFP plasmid was a gift from Prof. Yaron Shav-Tal (Bar Ilan University). MCP plasmid used for axonal live-imaging was phage-ubc-nls-ha-tdMCP-gfp (Addgene plasmid # 40649).
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