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

Abstract

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.

Molecular characterization of a stably transformed Bombyx mori cell line: identification of alternative transcriptional initiation sites of the A3 cytoplasmic actin gene

“…Our results are also of interest in light of the recent description of an insect expression system which utilizes the silkworm A3 cytoplasmic actin promoter and a combined transcriptional activation scheme (Lu et al 1997). The data presented also extend the ®ndings of McLachlin and Miller (1997) that the pac gene can be used as an eective selectable marker gene to establish stably transformed insect cell lines.…”

Cutting Edge: Dendritic Epidermal γδ T Cell Ligands Are Rapidly and Locally Expressed by Keratinocytes following Cutaneous Wounding

“…DETC or G8 TCR were transfected with pHSP70PLpac (9) into Drosophila melanogaster SC2 cells and selected with puromycin (Sigma). Protein expression, purification and biotinylation were conducted as described (7, 8).…”

Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus.

“…The locations of BmNPV homologous regions (hr) are also indicated. Names of ORFs were taken from Possee & Rohrmann (1997), Beniya et al (1998), Roncarati & KnebelMorsdorf (1997), McLachlin & Miller (1997), Olszewski & Miller (1997) or Rapp et al (1998) or the references listed (see footnote). The position of each ORF defines the A of the initiation codon (ATG) and the T of the termination codon (TAA\TAG\TGA) of its encoding strand.…”