Lr reaction

Manufactured by Thermo Fisher Scientific

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The LR reaction is a laboratory tool used for the rapid and efficient cloning of DNA fragments. It facilitates the directional insertion of a DNA insert into a destination vector, enabling the creation of recombinant plasmids. The LR reaction is a key component in various molecular biology workflows, such as gene expression studies and protein production.

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LR recombination reaction (38 citations) Gateway LR recombination reaction (25 citations) LR Clonase II reaction (19 citations) LR reaction mix II (7 citations) LR clonase reaction mix (4 citations) LR cloning reaction (2 citations) BP and LR clonase reaction (2 citations) MultiSite Gateway LR recombination reaction (2 citations) LR Clonase Reaction Kit (2 citations) LR recombination reaction system (1 citations)

124 protocols using lr reaction

Construction and Validation of Fluorescent Protein Reporter Vectors

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The mCherry fragment was amplified using the mCherry-F and mCherry-R primers and then ligated to pMDC163 and digested with XbaI and SacI. Therefore, GUS was replaced with mCherry, and pMDC163-mCherry was generated. SUC2_pro-pDONR207 was ligated to pMDC163-mCherry using the LR reaction (11791-020; Invitrogen, Carlsbad, CA, USA) to obtain pMDC163-SUC2_pro-mCherry. AtABCG14_pro-pDONR207 was ligated to pMDC107 using the LR reaction (Invitrogen, Carlsbad, CA, USA) to generate pMDC107-ABCG14_pro-GFP. All primers are listed in Supplemental Table S2.
AtABCG14 was amplified using primers AtABCG14-P1 and AtABCG14-P2 using the cDNA of WT as the template. After digestion with XbaI and SacI, the fragment was ligated to pMDC163, digested again with XbaI and SacI, and the GUS gene was replaced with AtABCG14 to obtain the pMDC163-ABCG14 plasmid. 4CL_pro-pDONR207 and SUC2_pro-pDONR207 were ligated to pMDC163-ABCG14 using the LR reaction (Invitrogen, Carlsbad, CA, USA) to form the pMDC163-4CL_pro-ABCG14 and pMDC163-SUC2_pro-ABCG14 plasmids. The plasmids were transformed in Agrobacterium tumefaciens strain GV3101 and infiltrated into the atabcg14 mutant using the floral dip method (Zhang et al., 2006 (link)). All primers used are listed in Supplemental Table S2. The promoter regions of AtABCG14, SUC2, and 4CL1 were 1,295, 2,129, and 1,001 bp in length, respectively.

Zhao J., Ding B., Zhu E., Deng X., Zhang M., Zhang P., Wang L., Dai Y., Xiao S., Zhang C., Liu C.J, & Zhang K. (2021). Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins. Plant Physiology, 186(4), 2111-2123.

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Yeast Two-Hybrid Screening of Transcriptional Regulators

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For a bait plasmid, the LATE and LATEΔEAR sequences in the pDONR207 vector were transferred into the pDEST_GBKT7 vector harboring the GATEWAY cassette (attR1-ccdB/Cm^r-attR2) between GAL4 DNA-BD and the ADH1 terminator of pGBKT7 (Chrontech) by the LR reaction (Thermo Fisher Scientific). These bait vectors were transformed into yeast strain Y2H gold and selected using SD medium lacking tryptophan (-W). Positive clones were confirmed by PCR using a primer set (Supplementary Table S1). Full-length coding sequences of KNU, CRC, TPL, and HDA19 were amplified by PCR with specific primer sets (Supplementary Table S1) and cloned into pDONR207 by the BP reaction (Thermo Fisher Scientific). For prey plasmids, KNU, CRC, TPL, and HDA19 sequences in the pDONR207 vector were transferred into the pDEST_GADT7 vector by the LR reaction (Thermo Fisher Scientific). These prey plasmids or blank control plasmid (pDEST_GADT7) were transformed into the yeast harboring the bait plasmids and spotted onto SD medium lacking tryptophan and leucine (-W/-L) or SD medium lacking tryptophan, leucine, and histidine (-W/-L/-H) supplemented with 0, 0.05, or 0.1 mM 3-amino-1,2,4-triazole (3-AT). These yeasts were grown at 30°C in the dark for 7–10 days.

Nakano Y., Kawai M., Arai M, & Fujiwara S. (2023). Genome editing and molecular analyses of an Arabidopsis transcription factor, LATE FLOWERING. Plant Biotechnology, 40(4), 337-344.

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Inducible Expression of KCTD17 and Trichoplein in RPE1 Cells

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Tet-On RPE1 cell lines that expressed myc-KCTD17 or MBP-trichoplein-flag (WT or K50/57R) were established with the same procedure described previously29 (link)46 (link)47 (link). The rtTA-advanced segment and the tTS transcriptional silencer segment from pTet-On Advanced and pQC-tTS-IN (BD Clontech) were recombined into the retroviral vector pDEST-PQCXIP and pDEST-PQCXIN, respectively, by the LR reaction (Invitrogen) to generate PQCXIN-Tet-On ADV and PQCXIP-tTS. The Elongation factor 1 alpha promoter (EF) in CSII-EF-MCS (a gift from Hiroyuki Miyoshi, RIKEN BioResource Center, Tsukuba, Japan) was replaced with a Tet-responsive promoter (TRE-Tight) from pTRE-Tight (BD Clontech) followed by a modified RfA fragment (Invitrogen) to make a Tet-responsive lentivirus vector, CSII-TRE-Tight-RfA. Fusion cDNAs with siRNA-resistant KCTD17 and trichoplein were recombined into the lentiviral vector by the LR reaction (Invitrogen) to generate CSII-TRE-Tight-myc-KCTD17 and CSII-TRE-Tight-MBP-trichoplein-3xFLAG, respectively. For induction of myc-KCTD17 or MBP-trichoplein-flag, Tet-On RPE1 cells were treated with 30 or 100 ng ml⁻¹ doxycycline (Sigma-Aldrich), respectively.

Kasahara K., Kawakami Y., Kiyono T., Yonemura S., Kawamura Y., Era S., Matsuzaki F., Goshima N, & Inagaki M. (2014). Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nature Communications, 5, 5081.

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Generating Inducible Lentiviral Vectors

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Construction of the lentiviral vector plasmids CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1, and CSII-TRE-Tight-CDK4R24C was previously described [38 (link)]. In brief, the EF1a promoter in CSII-EF-RfA (a gift from Dr. H. Miyoshi, RIKEN) was replaced with a tetracycline-inducible promoter, TRE-Tight, from pTRE-Tight (Clontech, #631059) to generate CSII-TRE-Tight-RfA. Human cyclin D1, human mutant CDK4 (CDK4R24C: an INK4a-resistant form of CDK4), and hTERT were inserted into the entry vector via a BP reaction (Invitrogen, Carlsbad, CA). These segments were then recombined with CSII-TRE-Tight-RfA through an LR reaction (Invitrogen) to generate CSII-TRE-Tight-cyclin D1, CSII-TRE-Tight-CDK4R24C, and CSII-TRE-Tight-hTERT. The rtTA segment from pTet-Off Advanced (Clontech) was amplified by PCR, recombined with the donor vector pDONR221 via a BP reaction (Invitrogen) to generate pENTR221-Tet-Off, and then recombined with a lentiviral vector, CSII-CMV-RfA, through an LR reaction (Invitrogen) to generate CSII-CMV-Tet-Off. Recombinant lentiviruses with vesicular stomatitis virus G glycoprotein were produced as described previously [39 (link)]. Keratinocytes were inoculated with 5 × 10⁶ infectious units [IU] each of CSII-CMV-hTERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C lentiviruses in the presence of 4 μg/mL of polybrene.

Inoue M., Kajiwara K., Yamaguchi A., Kiyono T., Samura O., Akutsu H., Sago H., Okamoto A, & Umezawa A. (2019). Autonomous trisomic rescue of Down syndrome cells. Laboratory Investigation; a Journal of Technical Methods and Pathology, 99(6), 885-897.

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Gateway Cloning for Transgenic Flies

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Thirty-seven nanograms of the destination vector, pBPGUw, were combined with 37.5 ng of DNA carrying a PCR fragment cloned in the Gateway vector in a LR reaction (Invitrogen) and incubated overnight at room temperature. TAM1 cells (Invitrogen) were transformed with 2.5 μL of the LR reaction and plated. A single isolate from each reaction was picked into a 96-well Beckman Deepwell block, allowed to grow overnight at 37 °C, and DNA was prepared by using the PerfectPrep kit (5 PRIME). The constructs were verified by analysis of restriction enzyme digests. A second isolate was picked in cases where there was a discrepancy between the observed and expected results. DNA for injection was prepared from 7 mL of overnight culture for production of transgenic flies.

Arbel H., Basu S., Fisher W.W., Hammonds A.S., Wan K.H., Park S., Weiszmann R., Booth B.W., Keranen S.V., Henriquez C., Shams Solari O., Bickel P.J., Biggin M.D., Celniker S.E, & Brown J.B. (2018). Exploiting regulatory heterogeneity to systematically identify enhancers with high accuracy. Proceedings of the National Academy of Sciences of the United States of America, 116(3), 900-908.

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Lentiviral Vector Construction for Inducible Overexpression

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Construction of the lentiviral vector plasmids CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1, and CSII-TRE-Tight-CDK4^R24C was previously described^{20 (link)}. In brief, the EF1a promoter in CSII-EF-RfA was replaced with a tetracycline-inducible promoter, TRE-Tight, from pTRE-Tight (Clontech, Mountain View, CA) to generate CSII-TRE-Tight-RfA. Human cyclin D1, human mutant CDK4 (CDK4^R24C: an INK4a-resistant form of CDK4), and TERT were inserted into the entry vector via a BP reaction (Invitrogen, Carlsbad, CA). These segments were then recombined with CSII-TRE-Tight-RfA through an LR reaction (Invitrogen) to generate CSII-TRE-Tight-cyclin D1, CSII-TRE-Tight-CDK4^R24C, and CSII-TRE-Tight-TERT. The rtTA segment from pTet-Off Advanced (Clontech) was amplified by PCR, recombined with the donor vector pDONR221 via a BP reaction (Invitrogen) to generate pENTR221-Tet-Off, and then recombined with a lentiviral vector, CSII-CMV-RfA, through an LR reaction (Invitrogen) to generate CSII-CMV-Tet-Off. Recombinant lentiviruses with vesicular stomatitis virus G glycoprotein were produced as described previously^{21 (link)}. Primary hepatocytes were inoculated with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4^R24C lentiviruses at the multiplicity of infection of 3 to 10 in the presence of 4 µg/mL of polybrene.

Nishiwaki M., Toyoda M., Oishi Y., Ishida S., Horiuchi S.I., Makino-Itou H., Kimura T., Ohno S.I., Ohkura T., Enosawa S., Akutsu H., Nakazawa A., Kasahara M., Kiyono T, & Umezawa A. (2020). Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing. Scientific Reports, 10, 17503.

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Cloning and GUS Assay of ABCB21 Promoter

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The 0.625 kb promoter fragment of ABCB21 upstream of the start codon was cloned into pENTR/D-TOPO (Thermo Fisher Scientific) then transferred into the Gateway compatible vector pGWB3 (Nakagawa et al., 2007 (link)) by LR reaction (Thermo Fisher Scientific). Constructs were transformed into Col-0 via floral dip (Clough and Bent, 1998 (link)). For GUS staining, tissues were incubated in 90% acetone for 20 min on ice, then immersed in staining solution (50 mM sodium phosphate buffer (pH 7.0), 0.1% Triton X-100, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 1 mM X-gluc) and incubated in the dark at 37°C for 5 h, unless otherwise noted. Stained samples were cleared with 70% ethanol before imaging. For sectioning tissue was dehydrated in a series of tert-butanol (TBA) and embedded in Paraplast Plus. Twenty micrometer sections were prepared using a Leica Reichert-Jung 2030 rotary microtome.

Jenness M.K., Carraro N., Pritchard C.A, & Murphy A.S. (2019). The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves. Frontiers in Plant Science, 10, 806.

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Cloning and Expression of Arabidopsis Proteins

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All the Arabidopsis proteins used in this study are listed in Supplemental Table 1. Full-length coding sequences of these proteins were cloned from a cDNA pool generated from 2-week old A. thaliana Col-0 ecotype plants by PCR-based Gateway BP cloning using the pDONR207 Donor vector (Thermo Fisher Scientific, Waltham, MA). Expression vectors for AP-MS and BiFC were constructed using the Gateway LR reaction with pK7FWG2 (Karimi et al., 2002 (link)), pDEST–^GWVYNE, and pDEST–^GWVYCE (Gehl et al., 2009 (link)). mCitrine and mCherry (Grünberg et al., 2013 (link)) were sub-cloned with the related gene into pDONR207 using the In-Fusion (Clontech) method, and then sub-cloned into pK7WG2 (Karimi et al., 2002 (link)) by means of LR reaction (Thermo Fisher Scientific) for the co-sublocalization assays (Supplemental Table 2).

Zhang Y., Chen M., Siemiatkowska B., Toleco M.R., Jing Y., Strotmann V., Zhang J., Stahl Y, & Fernie A.R. (2020). A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species. Plant Communications, 1(5), 100028.

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Transient Expression of Bacterial Effector in Plants

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R. solanacearum GMI1000 was grown on solid BG-11 medium plates or cultivated overnight in liquid BG-11 medium at 28°C (Morel et al., 2018b ). The ripE1 gene from R. solanacearum GMI1000 cloned in pDONR207 (donated by Nemo Peeters and Anne-Claire Cazale) was subcloned into pGWB505 by LR reaction (Thermo Fisher, USA) to generate a fusion protein with eGFP tag at the C terminus (Nakagawa et al., 2007 ). RipE1 and ripE1 mutants were inserted between BamHI and XhoI restriction sites on sXVE:GFPc:Bar estradiol-inducible vector using enzyme digestion (Schlücking et al., 2013 (link)). These generated binary vectors were transformed into Agrobacterium tumefaciens (Agrobacterium) GV3101 for transient or stable gene expression in N. benthamiana and A. thaliana plants. Agrobacterium carrying pGWB505 vectors were grown at 28°C and 220 rpm in Luria–Bertani medium supplemented with 50 mg/l rifampicin, 25 mg/l gentamycin, and 50 mg/l spectinomycin, while those carrying estradiol-inducible vectors were grown in 50 mg/l rifampicin, 25 mg/l gentamycin, and 50 mg/l kanamycin.

Sang Y., Yu W., Zhuang H., Wei Y., Derevnina L., Yu G., Luo J, & Macho A.P. (2020). Intra-strain Elicitation and Suppression of Plant Immunity by Ralstonia solanacearum Type-III Effectors in Nicotiana benthamiana. Plant Communications, 1(4), 100025.

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Generation of Transgenic Arabidopsis Lines

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The NahG gene was PCR amplified from pCIB200-NahG (Gaffney et al., 1993 (link)), cloned into pENTR/D-TOPO (Thermo Fisher Scientific), and recombined into pGWB14 (Nakagawa et al., 2007 (link)) by LR reaction (Thermo Fisher Scientific). pBIK1::BIK1-HA construct was described previously (Zhang et al., 2010 (link)). Binary vectors were then transformed into Agrobacterium tumefaciens strain C58C1, and transgenic lines were generated by floral dip (Clough and Bent, 1998 (link)). Transformants were selected for one-half-strength Murashige and Skoog medium supplemented with 15 μg ml⁻¹ Hygromycin B. For NahG-trangenic lines, expression of NahG-3×HA was confirmed by immunoblot in T3 homozygous lines. Horseradish peroxidase (HRP)-conjugated anti-HA antibody (Sigma) was used at a 1:1,000 concentration in conjunction with SuperSignal West Pico Chemiluminescent Substrate (Pierce) for detection. PR1 protein expression in NahG-transgenic lines was detected using anti-PR1 antibody (Wang et al., 2005 (link)). The PR1 mRNA abundance was measured by qPCR. PR1 protein expression, ROS, and bacterial growth assays were performed on sik1 NahG-HA line #1. MAPK activation was performed on sik1 NahG-HA lines #1 and #3. For BIK1-HA transgenic lines, expression of BIK1-HA protein was confirmed by western blot using an anti-HA antibody in independent T1 transgenic lines.

Zhang M., Chiang Y.H., Toruño T.Y., Lee D., Ma M., Liang X., Lal N.K., Lemos M., Lu Y.J., Ma S., Liu J., Brad D.a.y., Dinesh-Kumar S.P., Dehesh K., Dou D., Zhou J.M, & Coaker G. (2018). The MAP4 kinase SIK1 ensures robust extracellular ROS burst and anti-bacterial immunity in plants. Cell host & microbe, 24(3), 379-391.e5.

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