In fusion hd cloning plus

In-frame deletion plasmids were constructed by amplifying the flanking regions of specific genes by polymerase chain reaction (PCR) using Tks Gflex DNA Polymerase (TaKaRa Bio Inc., Kusatsu, Japan) and OH23 genomic DNA as the template according to the manufacturer’s instructions. Briefly, PCR amplification was performed using 30 PCR cycles consisting of denaturation at 98°C for 10 s, annealing at 55°C for 15 s, and elongation at 68°C for 1 min on a Bio-Red S1000 thermal cycler. The suicide vector pJQ200SK was digested with XbaI and BamHI (Thermo Fisher Scientific), and the target PCR fragments were ligated into the suicide vector using In-Fusion HD Cloning Plus (TaKaRa Bio Inc.). The recombinant vectors were transformed into E. coli DH5αλpir and confirmed using the universal primers M13F/M13R. The resulting plasmids were introduced into L. brunescens by conjugation. The deletion mutants ΔrpfF, ΔrpfC, and ΔrpfG were selected for double homologous recombination events because the suicide vector contained a sacB counter-selectable marker (Quandt and Hynes, 1993 (link)). Finally, all mutants were confirmed by PCR using specific primers (Table 2).

For HEK 293 cells transfections, full coding domain sequences for Siah1, Siah1ΔRING, CDHR1a, and CDHR1a LMA were amplified and cloned into pCIG2 using In-Fusion HD cloning Plus (Takara). Primers for Siah1 and Siah1DRING included a MYC tag while primers for CDHR1a and CDHR1a^LMA included a single FLAG tag. All constructs were verified using sanger sequencing. The HEK 293 cells were cultured at 37° in DMEM media until they 80% confluency and transfected using TransIT-LT1 Transfection Reagent (Mirus) at 37° for 24 h. Where indicated, treatment with 10 μM of MG132 for the last 4 h of transfection was performed. Co-IP was performed using lysates from cells treated with MG132 for 6 h prior to lysis. Western blotting and co-IP were performed as previously described (Piedade et al., 2019 (link)).

To clone the plin2 cDNA, tissue from the muscle and heart of adult casper zebrafish was dissected, pooled, and then RNA was isolated using the Zymogen Quick RNA Miniprep Kit (Zymo Research, Irvine, USA; catalog #R1054) according to manufacturer's instructions. The Invitrogen SuperScriptIII First-Strand Synthesis SuperMix Kit (Thermo Fisher, Waltham, USA; catalog #18080400) was used according to manufacturer's instructions to produce cDNA. CloneAmp HiFi PCR Premix (Takara, Mountain View, USA; catalog #639298) was used to PCR amplify the PLIN2 cDNA and gel purified via NucleoSpin Gel and PCR Clean Up (Takara, Mountain View, USA; catalog #740609.50). To generate pME-PLIN2-tdTOMATO, the PLIN2 cDNA was inserted on the 5’ end of pME-tdTOMATO using In-Fusion HD Cloning Plus (Takara, Mountain View, USA; catalog #638920). Gateway cloning using the Gateway LR Clonase Enzyme mix (Thermo Fisher, Waltham, USA; catalog #11791019) was employed to create the -3.5ubb:plin2-tdTomato construct with p5E-ubb, pME-PLIN2-tdTOMATO, and p3E-polyA into pDestTol2pA2-blastocidin (cells) (Heilmann et al., 2015 (link)) or pDestTol2CG2 (zebrafish) (Kwan et al., 2007 (link)).

The scFv sequence was obtained from plasmid pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE (Addgene #60906) (Tanenbaum et al., 2014 (link)). scFv-mNeonGreen-GB1-NLS was assembled using In-Fusion HD Cloning Plus multiple insert cloning (Takara Bio #638911) and integrated into the KpnI/SpeI sites of pCasper-attB-nos>tub3′UTR+1 kb (containing the nos promoter in EcoRI to direct maternal expression and tubulin 3′UTR+1 kb of downstream sequence in XbaI). To generate the noNLS version, the scFv-mNeonGreen-GB1 was amplified by PCR and inserted into KpnI/NotI of pCasper-attB-nos>tub3′UTR. ϕC31-based integration was used for specific re-integration of the transgene into sites 25C6 (chr2) and 86Fb (chr3) by the University of Cambridge microinjection service.

pMXs-HER2 vector was generated by cloning the HER2⁺ coding sequence (Addgene) into the pMXs-FLAG backbone using In-Fusion HD Cloning Plus (Takara Bio). HEK-293T cells (1.5 × 10⁶) were seeded into 6-cm dishes in 5 ml of complete medium. Twenty-eight hours later, 1 μg of pMXs-HER2 was mixed with 900 ng of retrovirus Pol/Gag, 150 ng of vesicular stomatitis virus G protein DNA, 130 μl of DMEM and 6 μl of 1 mg ml⁻¹ polyethylenimine. The mixture was incubated for 20 min at room temperature and added to HEK-293T cells dropwise. Eighteen hours later, the culture medium was replaced with 5 ml of DMEM supplemented with 30% heat-inactivated FBS and 1% P/S. Thirty hours later, the medium was collected and centrifuged at 1,000 r.p.m. for 5 min. The clarified supernatant was stored at −80 °C before infection. To establish stably expressing cell lines, 1.5 × 10⁶ cells were seeded in six-well plates in 2.8 ml of complete medium. Polybrene was added at 10 μg ml⁻¹, and cells were infected with 200 μl of virus-containing medium. Plates were centrifuged at 2,200 r.p.m. for 45 min and incubated at 37 °C and 5% CO₂. Twelve to 24 h later, cells were lifted with trypsin and plated in 10-cm dishes with 10 μg ml⁻¹ blasticidin S (Thermo Fisher Scientific). Stable cell lines were tested for expression of HER2 by flow cytometry.

OCLN’CR and Venus-OCLN expressing complemented cell lines were gifts of Matthew Evans, Icahn School of Medicine at Mount Sinai. To create OCLN expressing construct, OCLN ORF (template: OHu28110D, GenScript) was amplified using: forward primer (5’-GGA TCT ATT TCC GGT GAA TTC ATG TCA TCC AGG CCT CTT) and reverse primer (5’-ATC CGC GGC CGC TCT AGA CTA TGT TTT CTG TCT ATC ATA GTC TCC). pLVX vector was digested with EcoRI and XbaI. Amplified fragments were then inserted into digested pLVX with In-Fusion HD Cloning Plus (Takara) per the manufacturer’s instructions. To create constructs expressing OCLN mutants, Q5 Site-Directed Mutagenesis Kit (NEB) was used per the manufacturer’s instructions with pLVX OCLN as template and primer sets: 1) SS1 forward: 5’-AGT ACG ATA ATA GTG AGT GCT ATC C; reverse: 5’-TAA GAA GTA TCT TCT TGT TCT GG; 2) SS2 forward: 5’-AGA ACG AAG CAA GTG AAG GGA TC; reverse: 5’-ATT GAA TTC ATC AGC AGC AGC CA.