Simple piggyBac transposon-based mammalian cell expression system for inducible protein production

Abstract

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.glycoprotein | X-ray crystallography

Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting

“…The oligonucleotides were resuspended in RNAse-free water to a concentration of 100 μM. A VEGF-trap (aflibercept) mimic, a fusion protein of key domains from human VEGFR1 and VEGFR2 with human IgGFc, was made as described previously and shown to be effective for binding and neutralizing murine VEGF (36)(37)(38). Briefly, the gene encoding for VEGF-trap, with a carboxy-terminal histidine tag (H6), was cloned into a transposon-based system, and stable cell lines were derived after selection for neomycin resistance (Geneticin, 500 μg/ml).…”

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

“…These are heterogeneous populations of recombinant cells obtained by gene transfer and genetic selection but without subsequent cell cloning steps (Fan et al, 2013;Li et al, 2013;Ye et al, 2010). The main reasons for using cell pools over TGE are the need for only a small amount of plasmid DNA and the ease of volumetric scale-up by dilution (Fan et al, 2013;Li et al, 2013;Ye et al, 2010). Cell pools also have an advantage over clonal cell lines because of shorter and less costly development times (Ye et al, 2010).…”

“…Engineered transposable elements have been used to generate stably transfected CHO-and human embryonic kidney (HEK293)-derived cell lines for protein production (Alattia et al, 2013;Li et al, 2013;Matasci et al, 2011), and they have also served as gene transfer vectors for applications in gene therapy and gene discovery (Ding et al, 2005;Ivics et al, 1997;Mossine et al, 2013;Tsukiyama et al, 2011;Wu et al, 2006). piggyBac (PB), a class II transposable element originally derived from the cabbage looper moth, is one of the several transposons modified to function in mammalian cells (Fraser et al, 1996;Meir et al, 2011;Wilson et al, 2007).…”

“…PBase has been reported to be capable of mobilizing very large DNA molecules, such as bacterial artificial chromosomes (Li et al, 2011;Rostovskaya et al, 2012). However, the optimal transposon size may be below 14 kbp, since the frequency of transposition decreases as the size of the artificial transposon increases beyond this size (Alattia et al, 2013;Ding et al, 2005;Lacoste et al, 2009;Li et al, 2013;Meir et al, 2011;Wang et al, 2014).…”

Recombinant protein production from stable mammalian cell lines and pools

“…PB is the most widely used transposon for cell line generation and has been successfully applied to the overexpression of secreted and membrane proteins in CHO, CHO lec1, HEK293, and HEK293S-GnTI cells [22,[34][35][36][37]38 ]. Since transfection with the PB system yields a high percentage of recombinant cells, the generation of productive cell pools is feasible [4 ,37].…”