For retroviral expression of human PLK1 (gene ID: 5347), we used pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Plasmid #28289; Addgene, MA, USA). Retrovirus-based shRNA was prepared by EZ-10 Column DNA Purification Kit (Sangon Biotech) and retroviral particles were produced according to a previous method [45 (link), 46 (link)]. Fibroblast transduction was performed by incubating with the supernatant for 48 h. Puromycin was used to screen for expressing cells.
Prsi9 u6 sh ubic tagrfp 2a puro
Manufactured by Addgene
Sourced in United States
The PRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro is a plasmid construct designed for gene knockdown experiments. It contains a U6 promoter-driven shRNA expression cassette, followed by a TagRFP reporter gene and a puromycin resistance gene, all under the control of a UbiC promoter. This plasmid can be used for the expression of shRNA and the selection of transfected cells.
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3 protocols using prsi9 u6 sh ubic tagrfp 2a puro
1
Retroviral Expression of Human PLK1
2
Generating Lentiviral Overexpression Constructs
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All overexpression plasmids were based on the lentiviral vector pCW57-RFP-P2A-MCS (Plasmid #78933, Addgene). ΔPS CD97 was generated using primers against the CD97 isoform 3 expression vector and was assembled using Gibson Assembly. Short hairpin RNAs were cloned cloning into the pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Plasmid #28289, Addgene) shRNA expression vector.26 (link) Guide RNAs were designed using Benchling, selected based on off-target scores, and cloned into the LentiCRISPRV2-mCherry (Plasmid# 99154, Addgene) backbone. The MEKDD construct was in a pBabe-neomycin (Plasmid #1767, Addgene) expression vector.
3
Engineered Cell Lines for IAPP and MAPK1 Studies
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DBRAF-ER was cloned from pBabe-DBRAFER (a gift from Martin McMahon) by BamHI-SalI digestion and into Gateway entry vector pENTR1A by digestion with BamHI and XhoI. pLEG-DBRAFER-ires-Blast and control plasmid pLEG-mCherry-ires-Blast were generated by multisite gateway cloning with LR Clonase II Plus (12538120, Thermo Fisher Scientific) into pLEG(R1-R3) (Addgene, 48956; ref. 29) . DNA encoding shRNA to IAPP (shIAPP-1: sequence: 5 0 -ACCGGGCTGGTACTAAGAGGTTATTTgttaatattcatagcAAAT-AGCCTCTTAGTACCAGCTTTT) and MAPK1 (ERK2, sequence: 5 0 -ACCGGTATTACGACCTGAGTGATGAGgttaatattcatagcCTCG-TCACTCGGGTCGTAATATTTT) were PCR amplified (fwd: 5 0 -AT-ATATCTTGTGGAAAGGAAGACACACCGG, rev: 5 0 -ATATCTC-GTATGCCGTCTGAAGACTGCGAAAAAAc) and subcloned into pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Addgene, 28289) using BbsI. Bolded sequences are gene specific and the loop is in lower case. IAPP ORF was PCR amplified and cloned into the pENTR-D-TOPO. pLEG-IAPP ORF -ires-NAT and control vector pLEG-CFP-ires-NAT were made by Gateway cloning. NAT encodes N-Acetyl Transferase and confers resistance to the drug nourseothricin. Additional shRNAs in pLKO.1 and ORFs were obtained from McGill University High Throughput Screening Facility.
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