unc-119(ed3); sajIs50 [lin-26p::KAEDE + unc-119R] animals were generated by biolistic bombardment. The expression vector used to generate this transgene was made using MultiSite Gateway technology (Invitrogen). pCG150 destination vector (Addgene) was recombined with a pDONRP4P1R vector containing 5 kb of the lin-26 promoter (lin-26p; Martin et al., 2014 (link)), a pDONR201 containing the coding sequence for KAEDE protein amplified from Kaede-N1 (Addgene), and a pCM5.37 containing the unc-54 3′UTR (Addgene). This construct was used to generate transgenic animals by biolistic bombardment of unc-119(ed3) strain, using a PDS-1000/He system with the Hepta adaptor (Bio-Rad Laboratories) with the following parameters: 4-mm distance between the exits of the Hepta adaptor gas splitter and macrocarriers, 20-mm distance between stopping screens and target shelf, 1,350-psi rupture disks, and vacuum at 28 inches of mercury (Berezikov et al., 2004 (link)). Transgenic animals expressing AJM-1::GFP together with KAEDE were obtained through crossing unc-119(ed3); jcIs1 [ajm-1::GFP + unc-29(+) + rol-6(su1006)] IV hermaphrodites with unc-119(ed3); sajIs50 [lin-26p::KAEDE + unc-119R] males.
Hepta adaptor
Manufactured by Bio-Rad
Sourced in United States
The Hepta adaptor is a laboratory equipment product designed to connect multiple components within a laboratory setup. It provides seven connection ports, allowing users to establish multiple connections simultaneously. The core function of the Hepta adaptor is to facilitate the interconnection of various laboratory instruments and accessories.
Automatically generated - may contain errors
4 protocols using hepta adaptor
1
Generating Transgenic C. elegans Expressing KAEDE
2
Tobacco Plastid Transformation via Biolistic
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Plastid transformation was carried out using the biolistic protocol (26 (link)). Young leaves from aseptically grown tobacco plants were bombarded with vector DNA-coated 0.6 μm gold particles with a helium-driven particle gun (PDS-1000/He equipped with the Hepta adaptor; Bio-Rad). Primary spectinomycin-resistant lines were selected on an MS-based regeneration medium (26 (link)) containing 500 mg/l spectinomycin. For each construct, several independent transplastomic lines were selected and subjected to three additional rounds of regeneration on spectinomycin-containing medium to obtain homoplasmic plantlets.
3
Construction of hpk-1::GFP::H2B Reporter Strain
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A Phpk-1::GFP::H2B::hpk-1 3′ UTR expression construct was generated using the Gateway® Cloning System (Life Technologies). The putative promoter of the hpk-1 gene (corresponding to the region between nucleotide 2966 of cosmid W01H2 and nucleotide 36852 of cosmid F20B6) and the hpk-1 3′ UTR (corresponding to nucleotides 33865-32810 of cosmid F20B6) were amplified from N2 genomic DNA and the amplicons were incorporated into the Gateway pDONRP4-P1R and pDONRP2R-P3 vectors, respectively, in BP reactions to generate pSB009 and pMW008. A LR reaction was then performed with pSB009, a middle entry clone (pCM1.35 (GFP-H2B in pDONR201; Addgene plasmid 17248)), pMW008, and the destination plasmid pBCN40-R4R3 (Addgene plasmid 34915)56 (link). ~10 μg of the resulting plasmid pMW007 (Phpk-1::GFP::H2B::hpk-1 3′ UTR + Pmyo-2::mcherry) was introduced into young adult animals by biolistic transformation using the PDS-1000/He™ particle delivery system with a Hepta adaptor (BioRad, USA) according to the manufacturer’s instructions. A transgenic line was isolated using the dual antibiotic selection protocol57 (link) and backcrossed to wild type (N2) six times to generate strain HRN442.
4
Tobacco Plastid and Nuclear Transformation
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Tobacco plants for plastid transformation were grown under aseptic conditions on MS medium supplemented with 30 g/L sucrose until the seedling reached half the height of the Magenta box. Young leaves were collected and bombarded with the PDS1000/He particle delivery system equipped with a Hepta adaptor (Bio-Rad). Bombarded leaves were sliced into 5 mm × 5 mm pieces and placed onto RMOP (Regeneration Medium of Plants) medium containing 500 mg/L spectinomycin (50 (link)). For each construct, several independent transplastomic lines were subjected to additional rounds of regeneration on spectinomycin-containing medium to select for homoplasmy.
Nuclear transgenic plants were generated by Agrobacterium-mediated transformation (51 (link)). Leaves from 6-wk-old tobacco plants were infected with Agrobacterium tumefaciens (strain EHA105) containing vector pMT100. Infected explants were transferred to RMOP medium containing 100 mg/L kanamycin and 500 mg/L carbenicillin for selection. The hpRNA expression levels in nuclear transgenic tobacco plants were analyzed by qRT-PCR.
Rooted transplastomic and nuclear transgenic plants were transferred into soil and grown in a greenhouse for seed production.
Nuclear transgenic plants were generated by Agrobacterium-mediated transformation (51 (link)). Leaves from 6-wk-old tobacco plants were infected with Agrobacterium tumefaciens (strain EHA105) containing vector pMT100. Infected explants were transferred to RMOP medium containing 100 mg/L kanamycin and 500 mg/L carbenicillin for selection. The hpRNA expression levels in nuclear transgenic tobacco plants were analyzed by qRT-PCR.
Rooted transplastomic and nuclear transgenic plants were transferred into soil and grown in a greenhouse for seed production.
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