ComplexINC technology unlocks multigene delivery in primary cells

May 04, 2016

Incude pluripotent stem cells (iPS) were transduced with a virus encoding different cellular marker proteins. Oct4 was used as a marker for pluripotent stemm cells. All transduced cells express all heterologous proteins. Scale bar: 20 um.

Multigene delivery and subsequent cellular expression is a key technology for a wide range of applications in biology including structural research, cellular reprogramming and functional pharmaceutical screening. The construction of multigene circuits in mammalian cells is a core concept in synthetic biology and requires efficient delivery of complex heterologous DNA. For certain cell types including widely used HEK293 and HeLa cells, this can be achieved by plasmid-based transfection. However, a large number of cell lines and particularly primary cells are recalcitrant to plasmid transfection, thus requiring a different approach. Primary cells, however, are a central focus of current biological research efforts and multigene delivery in primary cells is highly desirable, but suitable tools were markedly lacking to date.

Researchers led by Philipp Berger (Paul Scherrer Institute, Villigen, Switzerland) and ComplexINC coordinator Imre Berger (University of Bristol, UK and EMBL) have now tackled this formidable challenge. In a publication in Nature Communications, they introduce MultiPrime, a baculovirus-based tool-kit for highly efficient multigene delivery in a wide range of mammalian cells including stem cells, fibroblasts, neurons and many others.

“Earlier, we had developed MultiLabel, a plasmid-based kit which performs well for transfecting mammalian cells with multiple genes” explains Philipp Berger. “We now wanted to expand our approach also to primary cells which are notoriously difficult to transfect”. The MultiPrime system consists of an easy-to-use assembly pipeline to put together many genes into large DNA constructions. These are then inserted into an engineered baculovirus optimized for mammalian cell transduction.

“Originally, baculovirus is specific to insect cells.” says Imre Berger. “We have effectively repurposed our time-tested MultiBac baculovirus now for mammalian cells such that it can enter also primary cells efficiently.” The MultiBac baculovirus is among the most successful systems for producing protein complexes in insect cells for structure determination, with over 1000 laboratories world-wide using MultiBac.

In the present publication, the authors demonstrate the aptitude of their novel MultiPrime system, for example for reprogramming fibroblasts into neurons and for producing functional antibodies. “We were ourselves surprised how well MultiPrime worked, even for zebrafish larvae” notes Maysam Mansouri, first author of the study. As a particular highlight, the study demonstrates the use of MultiPrime for CRISPR/Cas-9-based genome engineering, by inserting a marker gene into specific genomic sites in human chromosomes, with base pair precision.

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreements N° 279039 and N°613879.