Transfection of insect cell lines using polyethylenimine

Ogay, I. D.; Lihoradova, Olga; Azimova, Sh. S.; Abdukarimov, A; Slack, Jeffrey M.; Lynn, Dwight E.

doi:10.1007/s10616-006-9022-7

Cited by 46 publications

(38 citation statements)

References 31 publications

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“…This molecular mass had already been predicted from the amino acid sequence of hDPPIV (2,300 bp) (Tanaka et al 1992;Thoma et al 2003). The result was in consistence with previous studies (Chang et al 1999;Slack et al 2001;Slack and Lawrence 2002;Ogay et al 2006).…”

Section: Production Of Recombinant Baculovirussupporting

confidence: 89%

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

et al. 2013

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Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL -1 ) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P \ 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L -1 h -1 ).

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Section: Production Of Recombinant Baculovirussupporting

confidence: 89%

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

et al. 2013

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“…Trichoplusia ni BTI-TN-5B1-4 (Tn5), Spodoptera frugiperda IPLB-SF21AE (Sf21), and Sf21-specific clone (Sf9) cells were cultured in TNM-FH medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) at 27°C. The AcMNPV bacmid (AcBacmid) viruses were produced by transfection of Tn5 cells using polyethylenimine (PEI) (Polysciences Inc., Warrington, PA) with bacmid DNA extracted from Escherichia coli strain DH10Bac (Invitrogen) (33). BV was harvested by collection of culture medium, followed by centrifugation at 500 ϫ g for 5 min to remove cell debris.…”

Section: Methodsmentioning

confidence: 99%

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain

Garretson

McCoy

Cheng

2016

J Virol

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Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCEBaculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha-and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane be...

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“…Transfections were done with the transfection reagent, ExGen 500 (Fermentas) using methods adapted from Ogay et al (2006). Sf9 cells were seeded onto 6-well plates at a density of 1.25610 6 cells per well in 2 ml FBS/Grace's media.…”

Section: Methodsmentioning

confidence: 99%

“…These plasmids were previously used to study P74 localization in infected cells (Slack et al, 2001). Though designed as baculovirus shuttle vectors, pBAC-5-based plasmids are excellent for transient gene expression in insect cell culture (Ogay et al, 2006), due to the presence of an AcMNPV gp64 early/late promoter (Whitford et al, 1989;Blissard & Rohrmann, 1991). Homologous recombination of plasmids such as pBAC-5 with circularized viral DNA is an infrequent event (less than 1 %) (Kitts et al, 1990) and thus would not be significant for these transient experiments.…”

Section: Development Of a Transient Expression-based Assay For Characmentioning

confidence: 99%

“…After 2 h at 37 uC, viral DNA was extracted by phenol and chloroform/ isoamyl alcohol (24 : 1). The DNA was dialysed against TE at 4 uC in 10 K molecular weight cut-off Slide-A-Lyzer cassettes (Pierce).Transfections were done with the transfection reagent, ExGen 500 (Fermentas) using methods adapted from Ogay et al (2006). Sf9 cells were seeded onto 6-well plates at a density of 1.25610 6 cells per well in 2 ml FBS/Grace's media.…”

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confidence: 99%

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Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection

Slack

Lawrence²,

Krell

et al. 2008

Journal of General Virology

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Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin. INTRODUCTIONBaculoviruses are a group of arthropod-specific viruses (Zanotto et al., 1993) that have been applied as insecticides and as vectors for the expression of exogenous genes. They have a biphasic replication strategy producing two distinct viral phenotypes: the budded virion (BV) and the occlusion-derived virion (ODV). Both virion phenotypes have bilayer lipid envelopes surrounding bacillus-shaped nucleocapsids and contain double-stranded, circular DNA genomes. However, the integral protein composition of their envelopes and their roles in infection are distinct.BVs spread viral infection throughout host tissues by attaching to and entering host cells via receptor-mediated endocytosis (Volkman & Goldsmith, 1985). Endosomal acidification triggers envelope fusion with the endosomal membrane to release the viral nucleocapsid into the host cell (Leikina et al., 1992). In the case of group I alphabaculoviruses (Jehle et al., 2006), budding, attachment and envelope fusion are mediated by the viral protein GP64 (Blissard & Wenz, 1992) and for other baculovirus types these processes are mediated by an F protein (Pearson et al., 2000; Westenberg et al., 2002).ODVs are required in the horizontal transmission of baculoviruses between insect hosts (Kozlov et al., 1986). The ODVs of all baculoviruses are occluded into proteinaceous occlusion bodies (OBs) prior to release from the host (Rohrmann, 1986). The distinctive protein structure of OBs has a dual function. It serves to protect virions from deleterious environmental factors, and also acts as a delivery mechanism to transport the ODVs to the alkaline midgut where the cells are susceptible to infection.ODV envelopes are derived from the inner nuclear membrane (Braunagel & Summers, 1994) and contain envelope proteins that can survive the protease-rich environment of the insect midgut. ODVs attach to midgut columnar epithelial cells and fuse th...

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Transfection of insect cell lines using polyethylenimine

Cited by 46 publications

References 31 publications

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection

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