An RHV immunogen, coding for the nonstructural 3 (NS3) to NS5 region of RHV‐rn1 with GDD to AAG NS5B mutation, was synthesized using Mus musculus codons (GeneArt; ThermoFisher). The construct included a Kozak sequence, truncated shark invariant chain, and V5 epitope tag. SIi‐RHV‐NS3‐NS5mut‐V5 was cloned into a pENTR4 vector downstream of the human cytomegalovirus immediate early promoter and tetracycline operator. The coding cassette was moved to the ChAdOx1 destination vector using Thermo Fisher LR gateway cloning, and all steps were checked by sequencing. ChAdOx1 plasmids incorporating SIi‐RHV‐NS3‐NS5mut‐V5 linearized with PmeI were transfected into T‐REx‐293 cells (Thermo Fisher) for generation of viral‐vector vaccines. ChAdOx1‐SIi‐RHV‐NS3‐NS5mut‐V5 (ChAd‐NS) vaccines were manufactured by the Viral Vector Core Facility (Jenner Institute, University of Oxford). An identical process was followed for production of a ChAdOx1 vector encoding RHV antigen core, E1, E2, p7, and NS2 (ChAd‐S).
Gateway lr cloning
Manufactured by Thermo Fisher Scientific
Sourced in United States
Gateway LR cloning is a recombination-based cloning system that enables the transfer of DNA fragments between different vector systems. It facilitates the rapid and efficient cloning of genes, open reading frames, or other DNA sequences into a variety of expression and functional analysis vectors.
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Lab products found in correlation
Pdonr zeo, by Thermo Fisher Scientific (2 mentions)
Qiazol, by Qiagen (2 mentions)
Pdonr221, by Thermo Fisher Scientific (2 mentions)
Pentr directional topo cloning kit, by Thermo Fisher Scientific (2 mentions)
T rex 293 cells, by Thermo Fisher Scientific (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Csu x1, by Yokogawa (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Plan apo 40x objective, by Nikon (1 mentions)
Nis elements software 4, by Nikon (1 mentions)
Plx301, by Addgene (1 mentions)
Pdonr207, by Thermo Fisher Scientific (1 mentions)
Pdonr221 gateway vector, by Thermo Fisher Scientific (1 mentions)
Gblocks gene fragment, by Integrated DNA Technologies (1 mentions)
Gateway cloning method, by Thermo Fisher Scientific (1 mentions)
Ezchange site directed mutagenesis kit, by Enzynomics (1 mentions)
Multisite gateway lr recombination reaction, by Thermo Fisher Scientific (1 mentions)
Geneart gene synthesis service, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
22 protocols using gateway lr cloning
1
Construction of ChAdOx1-based RHV vaccines
2
Generation of CYFIP2 Rescue Constructs
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Alternative cyfip2 rescue constructs (ΔRac1; ΔFMRP) were generated from a pENTR cyfip2-EGFP plasmid [32 (link)] using custom primers and the Q5 Site Directed Mutagenesis Kit (NEB) to induce the desired C179R (ΔRac1) and K723E (ΔFMRP) mutations. Mutagenesis was confirmed using restriction digest and Sanger sequencing (Table S3 ). LR Gateway Cloning (ThermoFisher) was used to insert the altered cyfip2-EGFPs into the pDEST I-SceI hsp70 destination vector. Transgenic lines were created by microinjection into 1-cell stage embryos with a transgenesis mix containing phenol red, I-SceI enzyme, and the pDEST I-SceI hsp70:cyfip2-(C179R)-EGFP or pDEST I-SceI hsp70:cyfip2-(K723E)-EGFP plasmid.
3
Episomal Expression of Hc WET1 in G217B
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The Hc WET1 coding sequence, 1035 bp of the ACT1 promoter (ACT1p), and 729 bp of the CATB terminator (CATBt) were amplified from G217B gDNA and assembled into the Gateway entry vector pDONR/Zeo (Life Technologies) using restriction enzymes to generate BAS1504. A vector control construct, BAS252, was generated identically except lacking the WET1 CDS. Using LR Gateway cloning (Life Technologies) each pDONR/Zeo entry vector was recombined into the Hc episomal expression vector pDG33 (pDG33 is a derivative of pWU55 [72 (link)] with Hc URA5 added for selection and made Gateway compatible). The episomally-maintained vector control (ACT1p –CATBt) and ACT1p –WET1 –CATBt constructs were electroporated into G217Bura5Δ as previously described [72 (link)]. Protein and RNA was isolated simultaneously from each 37°C or RT culture using Qiazol (Qiagen, Netherlands) following the manufacturer’s instructions.
Cell morphology of vector control and WET1-expressing cells was determined using differential interference contrast (DIC) microscopy with a Yokogawa CSU-X1 (Yokogawa, Tokyo, Japan) spinning disk confocal mounted on a Nikon Eclipse Ti inverted microscope (Nikon, Tokyo, Japan) with a PLAN APO 40X objective (Nikon) and an Andor Clara digital camera (Andor, Belfast UK). Images were acquired by and processed in NIS-Elements software 4.10 (Nikon).
Cell morphology of vector control and WET1-expressing cells was determined using differential interference contrast (DIC) microscopy with a Yokogawa CSU-X1 (Yokogawa, Tokyo, Japan) spinning disk confocal mounted on a Nikon Eclipse Ti inverted microscope (Nikon, Tokyo, Japan) with a PLAN APO 40X objective (Nikon) and an Andor Clara digital camera (Andor, Belfast UK). Images were acquired by and processed in NIS-Elements software 4.10 (Nikon).
4
Versatile Episomal Expression Constructs
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2781 bp of the MS95 promoter/leader region (MS95p), 1045 bp of the GAPDH promoter (GAPDHp), and 729 bp of the CATB terminator (CATBt) were amplified from G217B gDNA and assembled into a Gateway entry vector pDONR/Zeo (Life Technologies) containing enhanced GFP (eGFP) using a CPEC cloning strategy [71 (link)]. This generated BAS1464 (MS95p –eGFP–CATBt) and BAS1514 (GAPDHp–eGFP–CATBt) constructs. All primer sequences are included in S4 Table .
G217Bura5Δ strains transformed with the GAPDHp and MS95p eGFP constructs were grown at 37°C to late log phase, diluted 1:25 into 5 mL HMM medium for growth at 37°C or 1:10 into 10 mL HMM medium for growth at RT. At 1 d, 2 d, and 3 d post-inoculation, cells were harvested by centrifugation and protein and RNA was isolated simultaneously from each 37°C or RT culture using Qiazol (Qiagen, Netherlands) following the manufacturer’s instructions.
G217Bura5Δ strains transformed with the GAPDHp and MS95p eGFP constructs were grown at 37°C to late log phase, diluted 1:25 into 5 mL HMM medium for growth at 37°C or 1:10 into 10 mL HMM medium for growth at RT. At 1 d, 2 d, and 3 d post-inoculation, cells were harvested by centrifugation and protein and RNA was isolated simultaneously from each 37°C or RT culture using Qiazol (Qiagen, Netherlands) following the manufacturer’s instructions.
5
Overexpression of Oncogenic RAS Variants
6
Gateway Cloning of E. coli ORFs
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Initial E. coli ORF primers were designed such that the 3' ends of the primers had homology to E. coli genes and 5’ ends contained a universal flanking sequence. A second round of PCR was performed with primers recognizing the universal flanking sequence and also having 5’ ends corresponding to gateway compatible attL1 (or attB1) and attL2 (or attB2) sequences on the forward and reverse primers, respectively. Resulting PCR products from attL sequence containing primers were directly cloned via gateway LR cloning (ThermoFisher Scientific) into yeast destination vector pAG416GPD-ccdB (Addgene) to create expression clones. PCR products from attB primers were subcloned via gateway BP cloning into vector pDONR221 (ThermoFisher Scientific) to create entry clones. These entry clones were then cloned via gateway LR to the pAG416GPD-ccdB destination vector to create expression clones. Some E. coli genes were synthesized as gBlocks from IDT and made gateway compatible by adding attL1 and attL2 sequences at the 5’ and 3’ ends, respectively, making them compatible for direct LR cloning to create expression vectors.
7
Genetic Constructs for Arabidopsis WUS and LEC1
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The coding regions of WUS and LEC1 and mutated WUS (WUSm2) were PCR-amplified from an Arabidopsis cDNA library with the appropriate primers (Table S1 ). To construct the WUSm1 constructs, mutated WUSm1 was separately amplified with two pairs of forward and reverse primers (At2g17950N and WUSm2R, WUSm2F, and 2g19750ox) (Table S1 ). The constructs for overexpression and SRDX fusion to the C-terminal and N-terminal regions for WUSm1, wild-type WUS, and LEC1 were based on the vectors named as pro35SG [35 (link)], pro35SSRDXG [36 (link)], and pro35S_M_SRDXG [3 (link)], respectively. After confirmation of the insert sequence, each transgene cassette was transferred into the binary destination vector pBCKH [36 (link)], which contained a hygromycin resistance gene, by Gateway LR cloning (Thermo Fisher Scientific, Waltham, MA, USA). For 35Spro:LEC1–VP16 construction, the LEC1 fragment flanked by Gateway attB1 and attB2 sequences (Thermo Fisher Scientific) at the 5′ and 3′ ends, respectively, was cloned into pDONR207 (Thermo Fisher Scientific) and introduced into pDEST_35S_VP16_HSP_GWB5 [37 (link)] (Fujiwara et al. 2014) by Gateway LR clonase II cloning (Thermo Fisher Scientific).
8
Generation of Zebrafish Transgenic Lines
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The pTol2-myo6b:CaSR-EGFP,cryaa:mCherry construct was generated by Gateway LR cloning (ThermoFisher) p5e myo6b,80 (link)pENTR CaSR-EGFP, and p3e MCS (AddGene # 7517481 (link)) into the destination vector pDESTtol2pACrymCherry (AddGene # 6402382 (link)) vector. DNA was midiprepped, phenol-chloroform extracted, and microinjected into one cell stage zebrafish embryos. Tol2 transgenesis was performed by microinjecting Tol2 mRNA and plasmid DNA as previously described.85 pTol2-hsp70:CaSR-EGFP,myl7:GFP was created by using Gateway cloning to insert p5e hsp70 and pENTR CaSR-EGFP into the pDestTol2CG2 backbone.79 (link)CaSR-EGFP was cloned into the pTol1-14xUAS:NTR-TagRFPT backbone using the In-Fusion HD Cloning Plus Kit (Takara Biosciences) to create pTol1-14xUAS:CaSR-EGFP. pTol1-14xUAS:CaSR-EGFP was microinjected into CaSRp190/+x TLF one cell stage larvae by microinjecting Tol1 mRNA and plasmid DNA as previously described.86 (link) Founders were identified by crossing injected g0 fish to GlyT2:Gal4,myl7:GFP45 (link) fish and screening for GFP expression in the brain.
9
Cloning and Transgenic Zebrafish Wnt9b
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The open reading frame of zebrafish wnt9b (ENSDARG00000037889) was amplified by PCR with AK3 and AK4 primers (Table 1 ) and cloned into the Gateway pDONR221 vector via Gateway BP cloning (Thermo Fisher Scientific) to generate pME-Wnt9b. The pDest_Tol2pA_hsp70l:wnt9b_IRES_EGFP vector was created by Gateway LR cloning (Thermo Fisher Scientific) from pDest_Tol2pA, p5E-hsp70l, pME-Wnt9b and p3E-IRES-eGFPpA. We injected 15 pg of plasmid into one-cell-stage embryos with 15 pg of Tol2 transposase mRNA.
10
Gateway Cloning of MCP Constructs
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The entry clone plasmid bearing the MCP coding sequence (pHCMM14) was combined with each of the destination vectors carrying distinct rBE candidates (see above) using Gateway LR cloning (Thermo Fisher Scientific, Cat # 11791020). Next, 1 μL of the reaction was transformed into E. coli and the correct clones were confirmed by Sanger sequencing. The resulting plasmids were then transiently transfected into human embryonic kidney 293 cells (HEK293XT, see below).
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