To generate transgenic UAS-hAdipoQ (Human Adiponectin), Adiponectin human cDNA (Origene, #TP315161) was PCR amplified and subcloned, using the Gateway cloning system (Invitrogen #K-2400-20), in Flag-tagged-UAS as destination vector (Carnegie Institution of Washington Vector collection). UAS-AdipoR-Myc was obtained from cloning a synthetic AdipoR cDNA (GeneSynthesis; GenScript) into a Myc-tagged-UAS as destination vector (Carnegie Institution of Washington Vector collection), using the Gateway cloning system (Invitrogen). A sequence of AdipoR corresponding to the cytoplasmic part of the receptor only (amino acids 1–201) was PCR amplified and subcloned in Myc-tagged-UAS as destination vector (Carnegie Institution of Washington Vector collection) to generate an activated form of AdipoR (AdipoR-act). grp78 cDNA was subcloned from FlyORF clone (DGRC #GEO03341) to an HA-tagged-UAS vector as destination vector using the Gateway cloning system (Invitrogen; Carnegie Institution of Washington Vector collection). Expression of the transgenes was validated by transfection in S2 cells and western blot. Constructs were then introduced into germ line by injections in the presence of the integrase (BestGene). (Primers sequences provided in Table S2 ; material available upon request).
Gateway cloning system
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Canada
The Gateway cloning system is a molecular biology tool used for the efficient and directional cloning of DNA sequences. It utilizes site-specific recombination to facilitate the transfer of DNA fragments between different vectors. The core function of the Gateway cloning system is to provide a versatile and streamlined approach for the manipulation and expression of genetic materials in various applications.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Polybrene, by Merck Group (7 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (5 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Pcdna dest40, by Thermo Fisher Scientific (4 mentions)
Polyethylenimine (pei), by Polysciences (4 mentions)
Blasticidin, by Thermo Fisher Scientific (4 mentions)
T4 dna ligase, by New England Biolabs (4 mentions)
Thermocycler, by Bio-Rad (4 mentions)
Hot start phusion polymerase, by Bio-Rad (4 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (3 mentions)
Pdonr221, by Thermo Fisher Scientific (3 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (3 mentions)
Pegfp n1, by Takara Bio (3 mentions)
Pdonr207, by Thermo Fisher Scientific (3 mentions)
Pinducer20, by Addgene (3 mentions)
Pentr d topo, by Thermo Fisher Scientific (3 mentions)
Plenti cmv neo dest, by Addgene (3 mentions)
Quickchange 2 xl site directed mutagenesis kit, by Agilent Technologies (3 mentions)
346 protocols using gateway cloning system
1
Generation of Transgenic Adiponectin Constructs
2
Generation of Transgenic Drosophila Constructs
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Transgenic constructs for injection were generated using the Gateway cloning system (Life Technologies). cDNAs were obtained by RT-PCR from ovarian or testes RNA of adult Drosophila melanogaster, Oregon R strain. mdAub, pdAub, pdAub-EXT, and AubPAZmut were generated by overlap PCR and inserted into the pENTR-D-TOPO directional cloning vector (Life Technologies). Transgenes were cloned into the pUASP-Gateway-phiC31 fly injection vector derived from the pCasPeR5-phiC31 vector containing GFP, mKate2, or Strep-FLAG tags using the Gateway cloning system (Life Technologies). The expression of each transgene was controlled using the yeast upstream activation sequence promoter (UASp) stably crossed with a maternal a-Tubulin67c-Gal4-VP16 (MaG4) driver. Transgenes were generated in flies by PhiC31-mediated transformation (BestGene) using PhiC31 landing pads on either chromosome two (BDSC #9736) or chromosome three (BDSC #9750). The GFP-wtAub and GFP-mdAub BAC line was generated by cloning the aub genomic locus from the BAC clone BACN04M10 into the pCasPeR4 vector using restriction sites XhoI and SpeI. Bacterial recombineering (Gene Bridges Counter Selection kit) was used to insert an in-frame GFP tag in the start site of Aub. GFP-wtAub and GFP-mdAub rescue lines were generated by crossing transgenic construct into the aub[HN]/ aub[QC] background.
3
TOPORS Interactome Mapping via Y2H
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The Y2H bait construct (pBD-TOPORS: GAL4 DNA binding domain (BD) fused with full-length TOPORS) was generated using the Gateway Cloning System (Life Technologies, CA, USA) according to the manufacturer’s instructions, using vectors, which had been modified from the original Stratagene (CA, USA) Y2H vectors to contain the att sequences, allowing for compatibility with the Gateway Cloning System. The candidate interacting partner of TOPORS, P26s4 encoded by the PSMC1 gene, was cloned in frame with the GAL4 activation domain (AD) using the Gateway Cloning System and the modified Stratagene vectors (pAD-PSMC1).
In-Fusion® Advantage PCR Cloning Kit (Clontech, CA, USA) was used to generate TOPORS deletion constructs, specifically pGBKT7-N-TOPORS (residues 1–380), pGBKT7-M-TOPORS (residues 373–781)and pGBKT7-C-TOPORS (residues 705–1045), for interaction characterisation in yeast.
The compatibility of the modified Stratagene and Clontech plasmid constructs was thoroughly validated prior to the Y2H screens.
In-Fusion® Advantage PCR Cloning Kit (Clontech, CA, USA) was used to generate TOPORS deletion constructs, specifically pGBKT7-N-TOPORS (residues 1–380), pGBKT7-M-TOPORS (residues 373–781)and pGBKT7-C-TOPORS (residues 705–1045), for interaction characterisation in yeast.
The compatibility of the modified Stratagene and Clontech plasmid constructs was thoroughly validated prior to the Y2H screens.
4
Cloning and Expression of Plant Genes
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The full-length cDNAs of CaKAN3, CaKAN4 and CaHSFA1,CaHSF8 were amplified by PCR using specific primer pairs (Supplementary Table 1 ) and then cloned into the entry vector pDONR207 by BP reaction using a Gateway cloning system (Invitrogen, 11789020). To construct the CaKAN3 or CaHSF8 destination vectors for the overexpression assay or prokaryotic expression, the full-length CaKAN3 and CaHSF8 genes were cloned into pEarleyGate plasmid vectors: pEarlyGate101, pEarlyGate103, pEarlyGate201, pEarlyGate20299 (link); pSPYCE, pSPYNE100 (link); pPGCL, pPGNL101 (link), pDEST-17 (Invitrogen, 11803012) or pDEST-15 (Invitrogen, 11802014) by LR reaction.
To construct the vectors for the VIGS assay, the specific gene fragments of CaKAN3, CaHSF8 or NLRs in their 3’UTRs to avoid the possible silencing of their homologous genes were amplified by PCR using specific primer pairs (Supplementary Table1 ), and each fragment was cloned into the entry vector pDONR207 by BP reaction individually and then into the destination vector pPYL279 by LR reaction using the Gateway cloning system (Invitrogen, 11791020).
To construct the vectors for the VIGS assay, the specific gene fragments of CaKAN3, CaHSF8 or NLRs in their 3’UTRs to avoid the possible silencing of their homologous genes were amplified by PCR using specific primer pairs (Supplementary Table
5
Cloning and Characterization of Populus Rab GTPases
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The full-length CDS of PtRabA1c, RabB1c, RabC1c, RabD2a, RabF2c, RabG3c and RabH1c were amplified from the cDNA of P. trichocarpa and was introduced into the pDNOR222.1 (Life technologies, Carlsbad, California, U.S.) to produce pENTR vectors. Subsequently, the genes in the pENTR vectors were validated by sequencing and then subcloned into pEarleyGate104 (ABRC stock DB3–686) to produce 35S::YFP-PtRabs constructs using the Gateway cloning system (Invitrogen). To generate Q74L substitution of PtRabE1b, site-directed mutagenesis was conducted by overlapping PCR. Four primers containing the desired changes (underlined) were used: PtRabE1b-QL-1, 5’-ATGGCTGCACCGCCAG-3′; PtRabE1b-QL-2, 5’-AGTTCGGAAACGTTCCA T
6
Genetic Engineering of Poplar Auxin Pathway
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The coding sequences of PtrFBL1, 2, 3, 4, 5, 6, 7, and 8, without stop codons, were amplified from the cDNA of hybrid poplar 84K (Populus alba × Populus glandulosa) and inserted into pEarleyGate101 (ABRC stock DB3-683) to produce the 35S::PtrFBL1-YFP, 35S::PtrFBL2-YFP, 35S::PtrFBL3-YFP, 35S::PtrFBL4-YFP, 35S::PtrFBL5-YFP, 35S::PtrFBL6-YFP, 35S::PtrFBL7-YFP, and 35S::PtrFBL8-YFP constructs, respectively, using the Gateway cloning system (Invitrogen). The approximately 2.5 kb 5′-UTR fragments of PtrFBL1, PtrFBL4, PtrFBL5, and PtrFBL7 were amplified from the genomic DNA of P. trichocarpa Torr. The primer sequences for the promoters are listed in Table S1 . The promoter fragments were then inserted into pDNOR222.1 and subcloned into pMDC164 to produce PPtrFBL1::GUS, PPtrFBL4::GUS, PPtrFBL5::GUS, and PPtrFBL7::GUS constructs using the Gateway cloning system (Invitrogen). The coding sequence of PtrFBL1 was amplified from the cDNA of 84K, cloned into pDNOR222.1 and sequenced. PtrFBL1 cDNA was further cloned into pMDC32 to produce 35S::PtrFBL1 constructs for transformation into poplar 84K. The transgenic poplar plants containing the auxin responsive promoter DR5 were obtained previously (Liu et al., 2014a (link)). At least five independent transgenic lines were used for further analyses.
7
Expression of EGFP-tagged CDS and Hrp48 constructs
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The CDS of Bruno FL (1-604) and ΔN (147-604) were tagged at the C-terminus with mEGFP using the Gateway cloning system (Invitrogen) in an intermediate pActin5c vector. Subsequently the EGFP-tagged sequence was sub-cloned into the UASp-attB vector and injected into VK-33 (y[1] M{vas-int.Dm}ZH-2A w[∗]; PBac{y[+]-attP-3B}VK00033) embryos for site-specific insertion into the attP landing site on chromosome III. Selection for positive transformants was based on eye color (mini-white gene). The transgene was balanced with Tm3Ser and the PhiC31 integrase was crossed out to obtain the final stocks. For Hrp48 truncations, EGFP tagging was done at the N terminus of FL and ΔC (1-205) using the Gateway cloning system (Invitrogen) in a pActin5c vector and then sub-cloned into the UASp-attB vector. VK-18 (vas-phi-ZH2A, PBac{y[+]-attP-9A}VK00018) embryos were injected for site-specific insertion into the attP landing site on chromosome II. The transgenes were balanced with CyO.
8
Overexpression and Knockdown of SWP73 in Arabidopsis
9
Construction of C9orf72 Constructs using Gateway
10
APOBEC3 Protein Expression Constructs
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APOBEC3A and APOBEC3B cDNAs were synthesized by GenScript with a beta-globin intron and a Flag tag in C-terminus. The plasmids expressing APOBEC3A-GFP/Flag, APOBEC3B-GFP/Flag, APOBEC3B-ΔCTD-GFP/Flag (amino acids 1–185), and APOBEC3B-ΔNTD-GFP/Flag (amino acids 186–382) were generated by inserting the cDNA into the pcDNA-DEST53 vector using the Gateway Cloning System (Thermo Fisher Scientific). The plasmids expressing APOBEC3B-Flag or APOBEC3B-E255Q-Flag were generated by inserting the cDNA into the pInducer20 vector using the Gateway Cloning System (Thermo Fisher Scientific). pcDNA3.1(+)-hA3Bi-3xHA was generated by overlapping PCR placing the human gamma globin intron (HBG2; accession M91037.1) between the natural boundaries of A3B exon 5 and exon 6 followed by ligation into a HindIII/XhoI digested pcDNA3.1(+) backbone upstream of in-frame carboxy-terminal 3xHA tags. All the APOBEC3B mutants were constructed by site-directed mutagenesis.
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