Efficient Transfection of Insect Cells with Baculovirus DNA Using Electroporation

Mann, Susan G.; King, Linda A.

doi:10.1099/0022-1317-70-12-3501

Cited by 27 publications

(7 citation statements)

References 20 publications

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“…CaM-PDE Expression in Baculovirus-Recombinant baculovirus were prepared using established protocols (28,30). Specifically, Sf9 cells were cultured in complete medium (composition: 46.26 g/liter Grace's insect medium, 0.35 g/liter NaHCO 3 , 3.3 g/liter lactalbumin hydrolysate, 3.3 g/liter yeastolate extract, 50 g/ml gentamycin, and 10% fetal bovine serum) until approximately 50% confluent.…”

Section: Bovine Lung Cdna Library Screening-approximately 10mentioning

confidence: 99%

Identification of Inhibitory and Calmodulin-binding Domains of the PDE1A1 and PDE1A2 Calmodulin-stimulated Cyclic Nucleotide Phosphodiesterases

Sonnenburg

Seger

Kwak

et al. 1995

Journal of Biological Chemistry

132

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Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca 2؉ /CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (K CaM ) of recombinant PDE1A1 (0.3 nM) was Ϸ10-fold less than the K CaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46 -106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaMbinding domain (residues 4 -46) produced a polypeptide that was still activated 3-fold by CaM (K CaM Ϸ 3 nM). However, complete CaM-independent activation occurred when residues 4 -98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76 -149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114 -137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca 2؉ concentrations is located between the two CaM-binding domains.Calmodulin-stimulated cyclic nucleotide phosphodiesterases (CaM-PDEs) 1 constitute a genetically diverse class of cAMP and cGMP hydrolyzing activities that are activated by calcium and calmodulin (1). Several different CaM-PDE activities have been identified (2). These CaM-PDE isozymes are distributed among discrete cell types in various tissues and have varying substrate specificity, specific activities, and activation constants (K CaM ) for CaM (2, 3). In cells that express CaM-PDEs, hormones that increase cytosolic Ca 2ϩ would be predicted to activate this isozyme, thereby decreasing cyclic nucleotide accumulation in response to hormones that stimulate cAMP or cGMP synthesis.In addition to Ca 2ϩ /CaM, certain CaM-PDEs may be subject to a secondary form of regulation via phosphorylation. In vitro, the 59-kDa (PDE1A1) and 61-kDa (PDE1A2) CaM-PDEs 2 are phosphorylated by cAMP-dependent protein kinase (4, 5), while the autophosphorylated form of CaM-dependent protein kinase II catalyzes the phosphorylation of the 63-kDa (PDE1B1) CaM-PDE (6). Phosphorylation increases the K CaM , effectively rendering these isozymes less sensitive to activation by Ca 2ϩ (7,8). In vivo, CaM-PDE phosphorylation would likely result in potentiation of cAMP or cGMP accumulation in response to hormonal stimuli involving coin...

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Section: Bovine Lung Cdna Library Screening-approximately 10mentioning

confidence: 99%

Identification of Inhibitory and Calmodulin-binding Domains of the PDE1A1 and PDE1A2 Calmodulin-stimulated Cyclic Nucleotide Phosphodiesterases

Sonnenburg

Seger

Kwak

et al. 1995

Journal of Biological Chemistry

132

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“…Electroporation has been used for delivery of DNA into the Helicoverpa zea and Musca insect embryos (Leopold et al 1996) and in a S. frugiperda cell line with Autographa californica nucleopolyhedrovirus (AcMNPV) DNA. This method yielded 100 times more progeny recombinant virus when compared to the calcium phosphate precipitation method (Mann and King 1989).…”

Section: Introductionmentioning

confidence: 99%

Transfection of insect cell lines using polyethylenimine

et al. 2006

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Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85-90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

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“…The modification typically employed for transfection of Drosophila cell lines (DINoCERA and DAWID 1983) is much less effective for lepidopteran cells. Electroporation is very effective for a S. Jrugiperda cells in particular (MANN and KING 1989) and other lepidopteran cell lines in general (M. J. Lipofection (BRL) has also been used (SHIV et al 1991), and in our hands this procedure is effective but has not represented a significant improvement over CaP04 technique (M. J. Fraser, unpublished).…”

Section: Practical Considerationsmentioning

confidence: 88%

Viral Expression Vectors

Muzyczka¹

1992

Current Topics in Microbiology and Immunology

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Efficient Transfection of Insect Cells with Baculovirus DNA Using Electroporation

Cited by 27 publications

References 20 publications

Identification of Inhibitory and Calmodulin-binding Domains of the PDE1A1 and PDE1A2 Calmodulin-stimulated Cyclic Nucleotide Phosphodiesterases

Identification of Inhibitory and Calmodulin-binding Domains of the PDE1A1 and PDE1A2 Calmodulin-stimulated Cyclic Nucleotide Phosphodiesterases

Transfection of insect cell lines using polyethylenimine

Viral Expression Vectors

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