MultiBacMam

BacMam is an increasingly popular system that facilitates the delivery of heterologous DNAs to a broad range of mammalian transduction targets. BacMam uses baculovirus as a vector and has distinct advantages over other mammalian transfection methods given the large heterologous DNA carrying capacity of baculovirus (>40kbp), the uptake of baculovirus by a broad range of mammalian cell types, and the sophisticated molecular biology technologies that have evolved over the last 20 years to support the generation of baculovirus expression vectors.

MultiBacMamis the first BacMam system designed specifically to allow modularly, DNA recombination-based assembly of multi-expression cassettes for multiprotein co-expression in mammalian cells.  MultiBacMam uses the same DNA recombination technologies as MultiBac, but with mammalian promoters, and a “mammalianized” baculovirus- displaying a VSV peptide on the baculovirus surface that increases virus uptake by an order of magnitude, and a stably integrated fluorescent protein to simplify monitoring of experiments. MultiBacMam transfer vectors are fully synthetic, comprising only functional DNA, and as a result, are small and easy to handle.

The MultiBacMam baculovirus genome (DH10EMBacVSV) is under continuous development to remove harmful/non-essential genes (e.g. v-cath and chiA) and DNA recombination hotspots and contains an integrated fluorescent protein expression cassette to allow for ease of monitoring virus titer. The MultiBacMam virus particle displays the VSV peptide on its surface to increase the efficiency of entry into mammalian cells by around 1 order of magnitude compared to prior BacMam virus systems.

MultiBacMam baculovirus-mediated transduction. (a) MultiMam plasmid DNA modules (pACEMam1,2; MDC, pMDK, pMDS) 10 containing expression cassettes under mammalian-active promoter control are combined into plasmid fusions by using the tandem recombineering (TR) method, and then integrated into the MultiBacMam baculoviral genome by Tn7 mediated transposition. Tn7L and Tn7R denote short DNA segments present on pACEMam1 and pACEMam2 which are recognized by the transposase. Ori stands for a regular origin of replication derived from pBR322, or R6Kγ denotes a conditional replication origin depending on a pair + background.

The attachment site (mini-attn7) is embedded in a LacZ gene enabling blue-white screening of positive integrands in E. coli cells harboring the baculoviral genome as a bacterial artificial chromosome (BAC). Kan R stands for kanamycin resistance marker. Sf21, Sf9, and Hi5 denote commonly used insect cell lines. MultiBacMam contains expression cassettes encoding for mCherry and vesicular stomatitis virus glycoprotein (VSV-G) integrated in the viral backbone. The composite MultiBacMam virus is used to infect insect cells for baculovirus production and amplification. VSV-G and mCherry are under baculoviral promoter control and thus expressed in insect cells.

Virus performance can be followed by eye due to the red color caused by mCherry. The high-quality MultiBacMam virus is then used to transduce mammalian cells. Virion image is adapted from drawing kindly provided by Kari Airenne 40. (b) Efficacy of plasmid-based transfection compared to MultiBacMam-mediated transduction demonstrating the superior MultiBacMam approach. U2OS cells were transfected or transduced with the construct VN-CDK5(D144N)-VC-p25. BiFC signal is in green and Hoechst 33342 staining is shown in blue. Scale bar, 50 µm

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