Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

Abstract

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity.

Functional characterization of the transcriptional regulatory elements of three highly expressed constitutive genes in the jewel wasp, Nasonia vitripennis

“…Each of the three cloned Nasonia TRE sequences was linked with enhanced GFP (EGFP) as a visible reporter and then cloned into pDP9e (Shirk & Furlong, ) for functional assessment using somatic transformation. The pDP9e vector contains a hr5IE1DsRed fluorescent reporter cassette for establishment of transfection efficiency and comparison of expression levels.…”

“…Once cloned into pGem‐T Easy, the EGFP fragment ( Bam HI‐ Ssp I fragment) from pEGFP‐N1 (TaKaRa, Mountain View, CA, USA) was cloned into the Bam HI‐ Zra I site 3′ of each of the three TRE sequences to produce pGem‐NVhsp70EGFP, pGem‐NVlsdpEGFP and pGem‐NVhsp90AEGFP. Each of these plasmids was restricted with Eco RI, filled with T4 DNA polymerase (NEB) according to the manufacturer's instructions, re‐restricted with Apa I and the NvTRE‐EGFP cassette was cloned into the ApaI‐BstZ17I site of pDP9e (Shirk & Furlong, ) to produce the somatic transformation vectors pDP9e‐NVhsp70EGFP, pDP9e‐NVlsdpEGFP and pDP9e‐NVhsp90AEGFP. Each of these plasmids was restricted with Kas I‐ Nde I to remove the NvTRE‐EGFP/hr5IE1DsRed cassettes, which were then cloned into the Kas I‐ Nde I of the LitpB1 piggyBac vector (Shirk & Furlong, ) to produce the germline transformation vectors LitpB‐NVhsp70EGFP/hr5IE1DsRed, LitpB‐NVlsdpEGFP/hr5IE1DsRed and LitpB‐NVhspEG90AEGFP/hr5IE1DsRed.…”

“…Each of these plasmids was restricted with Eco RI, filled with T4 DNA polymerase (NEB) according to the manufacturer's instructions, re‐restricted with Apa I and the NvTRE‐EGFP cassette was cloned into the ApaI‐BstZ17I site of pDP9e (Shirk & Furlong, ) to produce the somatic transformation vectors pDP9e‐NVhsp70EGFP, pDP9e‐NVlsdpEGFP and pDP9e‐NVhsp90AEGFP. Each of these plasmids was restricted with Kas I‐ Nde I to remove the NvTRE‐EGFP/hr5IE1DsRed cassettes, which were then cloned into the Kas I‐ Nde I of the LitpB1 piggyBac vector (Shirk & Furlong, ) to produce the germline transformation vectors LitpB‐NVhsp70EGFP/hr5IE1DsRed, LitpB‐NVlsdpEGFP/hr5IE1DsRed and LitpB‐NVhspEG90AEGFP/hr5IE1DsRed. The NVhsp70 TRE was replaced in the LitpB‐NVhsp70EGFP/hr5IE1DsRed vector with the D. melanogaster hsp70 promoter (Lis et al ., ) to form the LitpB‐Dmhsp70EGFP/hr5IE1DsRed vector.…”

A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

“…The main goal of our study was to develop a plasmid-based system for recovery of genetically modified DpCPV from cell cultures. Initially we tried to construct an insect cell line that stably expressed T7 polymerase, but due to the large size of the protein, we found that it could not be fully transcribed and expressed by several promoters (e.g., OpIE-1 [36], OpIE-2 [37], Drosophila metallothionein [38], Densoviral P9 [39]). Therefore, a helper virus like the recombinant vaccinia virus was adapted for expression of T7 polymerase.…”

N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae

“…The polyhedrin promoter is active during the late stage of baculoviral infection and has been used to express mammalian glycosyltransferases for the engineering of N -glycans in insect cells 10 . The actin promoter from B. mori is a constitutive promoter and has also been used for protein expression in insect cells 32, 33 . Only coexpression of P Pol -hGnT II and P Pol -hGalT I led to the expression of up to 35% biantennary terminally galactosylated N -glycans in silkworm pupae (Fig.…”