Baculovirus immediate early gene promoter based expression vectors for transient and stable transformation of insect cells

Vulsteke, Veerle; Janssen, Ine; Broeck, J. Vanden; Loot, AE; Huybrechts, Roger

doi:10.1111/j.1365-2583.1994.tb00139.x

Cited by 17 publications

(20 citation statements)

References 30 publications

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“…This efficiency is much better than that for Aedes nlbopietus C7-10 cells (0.007%; Gerenday et al, 1989), Aedes aeg~pti Mos20 cells (0.001-0.0001%; Lycett and Crampton, 1993), Spodoptera IPLB-SF-12 cells (0.26-0.59%; Helgen and Fallon, 1990), Bombyx Bin5 ceils (less than 2%; Vulsteke et al, 1993), and Drosophila $2 cells (5%; Vulsteke et al, 1993).…”

Section: Discussionmentioning

confidence: 85%

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Lan

Riddiford

1997

In Vitro Cell.Dev.Biol.-Animal

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The embryonic cell line, GV1, from Manduca sexta was transiently transfected with DNA constructs of the Drosophila hsp70 promoter fused to either a beta-galactosidase (pXH70ZT) or a chloramphenicol acetyl transferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell density, DNA:lipofectin ratio, and time of incubation were varied to determine the optimal conditions: 2 x 10(5) cells/ml, 1:3, and 5 h. Under these conditions, the transfection efficiency was about 40%. Heat inducibility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than that of pXH70ZT, containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in these cells. A 2 kb fragment of the proximal promoter region of the MHR3 gene containing a putative ecdysone response element was shown to be responsive to 20-hydroxyecdysone after its transfection into these cells.

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Section: Discussionmentioning

confidence: 85%

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Lan

Riddiford

1997

In Vitro Cell.Dev.Biol.-Animal

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“…To study the function of individual genes in the absence of viral infection, investigators have been interested in generating stably transformed insect cell lines which constitutively express introduced genes using promoters known to be active in lepidopteran ceil lines. Several genes have been expressed in this manner, including those encoding Escherichia colt [3-galaetosidase (Jarvis et al, 1990;Vulsteke et al, 1993), human tissue plasminogen activator (]arvis et al, 1990), AeMNPV p35 (Cartier et al, 1994) and a maize auxin-binding protein (Henderson et al, 1995). The seleetable marker genes used in all of the previous studies with insect cells have been either the bacterial gene for neomycin resistance (neo) or the hygromycin phosphotransferase (hygro) gene, which confer resistance to G418 sulfate and hygromycin B, respectiveb.…”

Section: Introductionmentioning

confidence: 97%

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

McLachlin

Miller

1997

In Vitro Cell.Dev.Biol.-Animal

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We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp 70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.

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“…Plasmid Construction and Stable Cell Line Establishment-The pBmIEGLacZ-BmIEGhyg, which encodes the hygromycin-resistant gene (26), was used to select stably transfected cells. All Rel protein expression vectors were derived from pRmHa-3 (27).…”

Section: Methodsmentioning

confidence: 99%

Interaction and Specificity of Rel-related Proteins in Regulating Drosophila Immunity Gene Expression

Han¹,

Ip²

1999

Journal of Biological Chemistry

104

106

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NF-B/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, and development. NF-B/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. We examined the activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. We also compared how different combinations of these transcriptional regulators control the activity of various immunity genes. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, the drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression.

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Baculovirus immediate early gene promoter based expression vectors for transient and stable transformation of insect cells

Cited by 17 publications

References 30 publications

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

Interaction and Specificity of Rel-related Proteins in Regulating Drosophila Immunity Gene Expression

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