Light it up: Highly efficient multigene delivery in mammalian cells

Abstract

Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics, and cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges. Here, we review recent multigene delivery and expression strategies and their exploitation in mammalian cells. We discuss applications in drug discovery assays, interaction studies, and biologics production, which may benefit in the future from our novel approach.

Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy

“…28 This combination imaging allows clear visualization of collagenrich bone structure and the interaction between HSPC clones and their microenvironment. Cohorts of animals were transplanted with the same population of transduced donor Lin Ϫ cells, and individual mice were killed at various time points, including 4,8,11,19,34, and 120 days after transplantation, to retrieve organs for imaging. The best-preserved and highest resolution imaging of large volumes of regenerating BM was obtained using whole mounts of bisected sternums.…”

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…Subsequently, efficient ACEMBL tool-kits have been developed also for multigene expression in eukaryotic hosts [34][35][36][37]. The integration of the ACEMBL technology in MultiBac, currently the lead technology for multiprotein complex production in insect cells, is described in a dedicated contribution of this issue [37].…”

“…Recombinant production offers solutions to these impediments, and a wide range of expression systems are available to produce proteins recombinantly in prokaryotic and eukaryotic hosts [31,33,[34][35][36][37][38][39][40][41][42][43][44][45]. Recombinant expression systems share in common that one or several DNA segments encoding for proteins, protein domains or multicomponent protein complexes are typically combined with DNA elements including DNAs that control transcription (promoters, terminators, others) and translation (ribosome binding sites, ShineDalgarno sequences, Kozak consensus sequences, enhancers, others) and inserted into a functional DNA module (plasmid, cosmid, artificial chromosome, genome, others).…”

“…6) in order to produce functional eukaryotic protein complexes requiring the authentic processing and post-translational machinery provided by eukaryotic hosts [31]. Multifusion plasmids generated from Cre-LoxP reactions are utilized by the ACEMBL-derived MultiMam system to facilitate simultaneous multigene introduction into mammalian cells [35,36] (see also 3.2). The MultiBac baculovirus / insect cell system has been upgraded for robotics by incorporating ACEMBL DNA modules (MIE and HE / BstXI sites) for automatable and theoretically unlimited multigene insertion into a baculoviral genome for protein co-expression in insect cells [34] (see also Chapter on MultiBac by Sari and co-workers in this issue).…”

“…We implemented TR to facilitate rapid generation of multicomponent gene expression circuits from Acceptors and Donors containing mammalian cell active promoters [35,36] ( Fig. 6).…”