Geneart site directed mutagenesis plus kit

Zebrafish Ikk1 (Chuk) cDNA was purchased from Open Biosystems (Clone ID: 5914247, GE Dharmacon). Full-length (FL)-Ikk1 was amplified with Advantage 2 Taq Polymerase (Clontech) using forward primer 5′-GCTAGCTGTCAATATGGAGAAACCCCCT-3′ and reverse primer 5′-TAATGGATTTGGTACCGACAAACGCGCTGATTTA-3′. The amplified PCR products were cloned into pCR Blunt II-TOPO using the Zero Blunt^® TOPO^® PCR Cloning Kit (Life Technologies). To generate tdTomato fusion constructs, pCRTOPO-FL-Ikk1 and ptdTomato-N1 vector (Clontech) were digested with KpnI and NheI, and ligated. The Ikk1(C179A)–tdTomato variant was generated from FL-Ikk1–tdTomato using site-directed mutagenesis (GeneArt^® Site-Directed Mutagenesis PLUS Kit, LifeTechnologies). The constructs were subsequently cloned into a plasmid containing the zebrafish tp63 promoter (pT2KXIG_tp63:AcGFP, courtesy of Gromoslav Smolen, Harvard University, Cambridge, MA). First, GFP was removed via digestion with BamHI and NotI, and both sites were Klenow blunt-ended. The Ikk1–tdTomato plasmids were digested with SnaBI and SfoI and ligated into pT2KXIG_tp63 to generate tp63:FL-ikk1–tdTomato and tp63:ikk1(C179A)–tdTomato. HEK001 were transfected using Nanofectin (PAA, Q050-005) when 60% confluent in 8-chamber vessels. It was determined that 0.5 µg of DNA per 0.6 µl of transfection reagent was optimal.

Human KL1-expression vector was sourced commercially (RC220944; OriGene Technologies, Rockville, MD, USA) and the KL1 protein was expressed in a pEGFP-C1 vector (Clontech, Mountain View, CA, USA) (GFP-KL1) as previously reported [20 (link)]. Mouse α-TAT1 expression vector was also purchased commercially (MR206707; OriGene Technologies) and flag-tagged α-TAT1 was expressed from a pCMV6-entry vector. The expression vector for rat katanin (pEGFP-C1-p60 katanin) was kind gift of Dr. PW. Baas, Drexel University, Philadelphia, PA. The human α-tubulin construct [20 (link)] was N-terminally GFP-tagged and expressed in pEGFP-C1 (GFP-tubulin(wt)). The tubulin K40R mutation was introduced using a GeneArt Site-Directed Mutagenesis PLUS kit (Life Technologies, Carlsbad, CA, USA) (GFP-tubulin(K40R)) and the structure of the construct was confirmed by sequencing.

Using rabbit HEV-3ra infectious clone LR or indicator replicon LRG as the backbone, the HEV-3ra mutants containing a single-site mutation, Y1320H, K1383N, K1634G, or K1634R, were constructed with the GeneArt site-directed mutagenesis system (Invitrogen-Thermo Fischer, Waltham, MA, USA) according to the manufacturer’s instructions. The LRG double mutant Y1320H/K1383N was constructed with GeneArt site-directed mutagenesis PLUS kit (Thermo Scientific, Waltham, MA, USA). As recommended for the mutagenesis system, PCRs were performed with AccuPrime Pfx DNA polymerase (Invitrogen-Thermo Scientific, Waltham, MA, USA). All primers used in this study for viral genomic sequencing and introduction of mutations are commercially synthesized (Integrated DNA Technologies, Coralville, IA, USA) and listed in Table S2.
One Shot MAX Efficiency DH5α-T1R competent cells (Invitrogen-Thermo Fischer, Waltham, MA, USA) were transformed with each of the HEV-3ra constructs containing specific mutation(s). The transformed cells were cultured in 50 mL lysogeny broth medium with ampicillin, and plasmid midipreps were performed with Qiagen Plasmid Plus midikit (Qiagen, Germantown, MD, USA). The entire viral genome in each of the HEV-3ra constructs was confirmed through Sanger sequencing to ensure that no additional nucleotide substitution had been introduced.

Mutation of the predicted 3′ UTR binding site of miR-29b-1/a on pEZX-MT06 miRNA 3′ UTR target dual luciferase expression vectors was performed using the GeneArt^® Site-Directed Mutagenesis PLUS Kit (ThermoFisher Scientific) according to manufacturer’s instructions. Where indicated, HEK-293 cells were plated in 24 well plates and co-transfected with 200 ng of wild type (wt) or mutant (mut) vectors plus a 20 nM final concentration of miR-29b-1/a mimic, anti-miR-29a or negative control mimic using FuGENE HD (Promega) according to manufacturer’s protocol. Twenty-four h post transfection, a dual luciferase assay (Promega) was performed. Luciferase expression was determined relative to negative control mimic and statistical evaluation performed using GraphPad Prism Software (Graph Pad Software, San Diego, CA).