To create the TOC1 overexpressing construct, TOC1 transcript was amplified from hybrid poplar genome using the primers TOC1_FW 5’- ATGGAGGGAGAGGTAGATGAGC-3’ and TOC1_RV 5’-TTAAGATCCTGAAGCATCGTCCTCAG-3’. The resulting piece was cloned along with 35 S promoter into dpGreen destination vector using the MultiSite Gateway Kit (Invitrogen, MA, United States). Empty construct used as control was assembled in the same manner without TOC1 sequence. In vitro poplar plantlets were transformed following the protocol previously reported34 (link), and leaf samples were collected after 2 days at the peak of FT2 expression.
Multisite gateway kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MultiSite Gateway Kit is a molecular biology tool designed for cloning and transfer of DNA fragments between multiple entry vectors and destination vectors. It facilitates the construction of complex plasmids or expression constructs by enabling the assembly of multiple DNA fragments in a single reaction. The kit provides the necessary components and protocols for performing this multi-part cloning process.
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Lab products found in correlation
4 protocols using multisite gateway kit
1
Overexpressing TOC1 in Hybrid Poplar
2
Generating Dominant-Negative pak2a Transgenic Lines
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The dominant-negative pak2a (pak283‒149) was generated by deleting CRIB motif, which retains the dimerization domain, the kinase inhibitory domain, and the inhibitory switch domain (Chu et al., 2004 (link)). MultiSite Gateway Kit (Invitrogen) was used to clone the hsp70 promoter and dnpak2a fragment into the pDESTol2pA2 vector. The hsp70–dnpak2a construct was co-injected with Tol2 transposase mRNA into one-cell stage embryos to generate transgenic lines.
3
Hepatocyte-specific mCherry Expression Zebrafish
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Tg[fabp10:mCherry] fish expressing mCherry exclusively in hepatocytes were generated using MultiSite Gateway™ kit (Thermo Fisher Scientific, Waltham, MA, USA) to produce vectors with Tol2 transposon sites45 (link). A 2.8-kb promoter of the fabp10 gene23 (link) was cloned into the p5E-mcs vector. Multisite Gateway cloning46 (link) was performed with the destination vector pDestTol2pA2, the 5′ entry vector containing the fabp10 promoter, the middle entry vector containing pME-mCherry, and the 3′ entry vector containing p3E-polyA. DNA constructs (25 pg) and Tol2 mRNA (25 pg) were injected into wild-type zebrafish embryos at the one-cell stage.
4
Generating Hepatocyte-Specific mCherry Zebrafish
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Tg[fabp10:mCherry] fish expressing mCherry exclusively in hepatocytes were generated using MultiSite Gateway™ kit (Thermo Fisher Scientific, Waltham, MA, USA) to produce vectors with Tol2 transposon sites 34 (link) . A 2.8-kb promoter of the fabp10 gene 21 (link) was cloned into the p5E-mcs vector. Multisite Gateway cloning 35 (link) was performed with the destination vector pDestTol2pA2, the 5' entry vector containing the fabp10 promoter, the middle entry vector containing pME-mCherry, and the 3' entry vector containing p3E-polyA. DNA constructs (25 pg) and Tol2 mRNA (25 pg) were injected into wild-type zebrafish embryos at the one-cell stage.
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