Development of a novel set of Gateway‐compatible vectors for live imaging in insect cells

Abstract

Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high-throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway-compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C-terminal or the N-terminal ends. Additionally, a collection of organelle-specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self-interaction of the MRCV viroplasm matrix protein P9-1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high-throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins.

AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

“…For the establishment of the Sf-9.RNu cell line, 1×10 6 cells growing in 35 mm-diameter culture dishes were transfected with 1 µg of plasmid pIB-LMNA-R [28] using Cellfectin II® reagent (Invitrogen). Recombinant cellular clones were selected using blasticidin, at concentration of 60 µg/ml.…”

“…To obtain Ac109 N-terminal or C-terminal fluorescence fusions, we recombined TOPO.109N entry vector containing the ac109 coding sequence without the ATG initiation codon or TOPO.109C entry vector containing the ac109 coding sequence lacking the STOP codon into pIB-YW or pIB-WY destination vectors [28] using the Gateway system (Invitrogen), giving rise to pIB-Y-109 and pIB-109-Y vectors respectively.…”

“…polyhedrin gene and its promoter were PCR amplified with primers PH-up 5′ GCCGGCATAGTACGC 3′ and POLSTOP [32] and cloned into pGEM-Teasy intermediate vector giving rise to pGEMpPOL. ac109 promoter sequence and ac109 gene without the STOP codon were amplified by PCR and cloned in pCR®8/GW/TOPO® to obtain TOPO.p109-109C entry vector, which was recombined into pIB-WY destination vector [28]. p109-109Y sequence was amplified with 109Pr and GFP-rev primers and cloned into pCR®2.1-TOPO® vector.…”

Infectious Bursal Disease Virus non-structural protein VP5 is not a transmembrane protein

“…CFP was obtained from vector pECFP (PT3258-5, Clontech) using XbaI, restriction enzyme and cloned into pcDNA-VP5 to obtain pVP5-CFP. YFP, without its stop codon, was obtained from pTgYFPH (Maroniche et al, 2011). YFP was excised with HindIII digestion and cloned into pcDNA-VP5 to obtain pYFP-VP5.…”

RNA interference‐mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus

“…Cursory domain analyses (hydrophobic amino terminus with Pro/Gly-rich region), homology with microsomal cytochrome P450s and subcellular localization prediction algorithms (WoLF PSORT and Target P1.1) suggest that LhSpot also localizes to the ER/secretory pathway. To provide further insights into the presumptive subcellular localization of LhSpot, we co-expressed fluorescent chimeras of LhSpot (LhSpot-Venus) and the human KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor (HsKDELR-mCherry), a marker of the ER/ secretory pathway (van der Vlies et al, 2002;Maroniche et al, 2011), in cultured Trichoplusia ni cells. Cells expressing LhSpot-Venus exhibited a diffuse green fluorescent pattern typical of ER that extensively overlaid with the HsKDELR-mCherry red fluorescent signal ( Fig.…”