Highly efficient baculovirus-mediated multigene delivery in primary cells

Abstract

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.

Figure 1. Multigene expression in primary cells by MultiPrime.

HUVEC, REF cells and rat cortical neurons were infected with a MultiPrime baculovirus encoding EBFP2-Nuc (labelling the nucleus), mTFP1-FYVE (PI-3-P containing endosomes), tubulin-EYFP (cytoskeleton), Mito-dsRed (mitochondria) and PLCδ-PH (PI-4,5-P2; plasma membrane). iPS cells were transduced with a virus encoding mTFP1-actin, EYFP-tubulin and Mito-dsRed. Oct4 was used as a marker for pluripotent stem cells. All infected cells express all heterologous proteins. Scale bar, 20 μm.

Figure 2. Infected cells retain functionality.

(a) COS7 cells were infected with MultiPrime baculoviruses expressing the indicated fluorescently-tagged RAB GTPases. Cells were stimulated for 3 h with Cy5-labelled EGF. As expected, EGF was found in RAB7A vesicles (arrows) and RAB5A vesicles (arrowheads) but not in RAB11 vesicles. A quantitative time-resolved analysis is provided (Supplementary Movie 1). (b) PAE cells stably expressing VEGFR2 and neuropilin-1 were infected with baculoviruses expressing the indicated fluorescently-tagged RAB GTPases. Cells were then stimulated with Nt647-labelled VEGF. VEGF can be found in RAB5 and Rab7 vesicles (arrows). (c) Tube formation: HUVEC were infected with a baculovirus expressing mTFP1-actin, EYFP-tubulin and Mito-dsRed (only red channel is shown). DAPI was used to counterstain nuclei of all cells. Approximately 30% of cells were infected. Infected and uninfected cells contribute to tubes. (d) Migration: HUVEC were infected with a baculovirus expressing mTFP1-actin, EYFP-tubulin and Mito-dsRed (only red channel is shown). Approximately 30% of cells were infected. Infected and uninfected cells migrate at same rates. Scale bar, 20 μm (a,b); 500 μm (c,d).

Figure 3. Transduction efficacy.

(a) Transduction by MultiPrime baculovirus was compared to plasmid-based transfection. MultiBacMam bacoluvirus expressing VSV-G and EMBacY baculovirus devoid of VSV-G were used. For plasmid-based transfection, the plasmid (13 kb) used originally inserted into the recombinant baculoviruses was utilized. MultiPrime-mediated transduction is markedly superior in all cell types tested. Data shows mean value±s.d.; n=3; ****P<0.0001 determined by comparing transfection with transduction with or without VSV-G using one way analysis of variance followed by the Dunnet's post hoc test. (b) Effects of the MOI are shown. (c) PAE cells were infected with a MultiPrime baculovirus expressing three proteins at a MOI of 500. DAPI was used to counterstain nuclei of all cells. Virtually all cells are infected and express all heterologous proteins. Scale bar, 100 μm. (d) Toxicity of transduction was measured by means of a MTT assay. MultiPrime transduction exhibits comparable, low toxicity as plasmid transfection. (e) The persistence of heterologous expression was quantified by fluorescence in REF cells. The percentage of positive cells for each individual protein is shown over time.

Figure 4. Modulation of expression levels with alternative promoters.

(a) Transduction of HEK, PAE and REF cells with baculoviruses expressing EYFP-tubulin under the control of the indicated promoters and citrine under the control of the CMV promoter is shown. (b) Quantification of the blots. EYFP-tubulin/citrine ratio was used as a measure for promoter strength. Endogenous tubulin was used as loading control. Data shows mean value±s.d.; n=3; ****P<0.0001, ***P<0.001 determined by comparing CMV promoter with alternative promoters using one-way analysis of variance followed by the Dunnet's post hoc test. The CMV promoter is the strongest promoter in all tested cell lines. (c) HeLa-tTA cells were infected with a baculovirus expressing EYFP-tubulin under the control of a tetracyclin inducible element and citrine under the control of the CMV promoter. (d) Quantification of the blots shown above. Data shows mean value±s.d.; n=3; **P<0.01 determined by comparing induced versus non-induced tet promoter using one-way analysis of variance followed by the Tukey post hoc test. No significant difference between CMV promoter and induced tet promoter.

Figure 5. Promoters active in mammalian and insect cells.

(a) Structure of the tested dual promoters is shown schematically. In CMVP10, the baculoviral very late promoter p10 was inserted downstream of the CMV promoter. In CMVintP10, the p10 promoter was placed within an intron and is spliced out from the transcript of the CMV promoter. Grey arrow: transcription initiation of the CMV promoter; black arrow: transcription initiation of the p10 promoter. (b) HEK293, PAE and REF cells were infected with a MultiPrime baculovirus expressing EYFP-tubulin under the control of the above promoters and citrine under the control of the CMV promoter. The lysates of the cells were analysed by western blotting. (c) Quantification of blots shown in b. EYFP-tubulin/citrine ratio was used as a measure for promoter strength. Endogenous tubulin was used as loading control. The CMVintP10 promoter expresses at a similar level as the original CMV promoter, while the CMVP10 promoter expresses at a lower level in mammalian cells. (d,e) The same baculoviruses were used to infect insect (Sf21) cells. Citrine was expressed by a P10 driven expression cassette in the backbone of the baculovirus. Data shows mean value±s.d.; n=3; there is no significant difference (P>0.05) by comparing the two CMVP10 promoter variants using the Student's t-test.

Figure 6. MultiPrime applications.

(a) Genome engineering. Infection of HUVEC with a baculovirus containing a HMGA1-EGFP homology construct did not lead to cells with HMGA1-EGFP expression (left). Co-expression with Cas9 and HMGA1-gRNA1 (2nd panel) or co-expression with Cas9, HMGA1-gRNA1 and HMGA1-gRNA2 (3rd panel) led to cells with HMGA1-EGFP expression in the nucleus. Correct integration of the homology construct was verified with PCR. The wild-type allele yielded a fragment of 2,023 bp, whereas the mutant allele results in a fragment with 1,139 bp (right). Scale bar, 50 μm. (b) Reprogramming of cells. MEF cells were infected with a Multiprime virus expressing Ascl1, Brn2 and Myt1L or co-infected with three lentiviruses individually expressing the same transcription factors. Both strategies led to cells with neuron-like morphology that express the neuronal markers MAP2 and β-tubulin III. Scale bar, 50 μm. (c) Functional antibody expression. MultiPrime viruses, encoding light and heavy chains of three different IgGs (anti-VEGF, anti-VEGFR2, and unspecific) were used to express antibodies in HEK293 (left) and also in insect cells (middle). HUVEC cells were infected with these viruses and the cells were used in a Matrigel-based angiogenesis assay. As expected, only the anti-VEGF antibody is capable of blocking tube formation Scale bar, 500 μm. Data shows mean value±s.d.; n=4; **P<0.01 when comparing VEGF function-blocking antibody versus control antibody. There is no significant difference (P>0.05) when comparing the non-function-blocking VEGFR2 antibody with the control antibody. Both P values are determined using one way analysis of variance followed by the Dunnet's post hoc test. (d) Baculovirus-mediated gene expression in zebrafish. Dorsal view of the head of a 3-day-old zebrafish larva after injection of MultiPrime baculoviruses expressing mTFP1-actin, EYFP-tubulin and Mito-DsRed into the hindbrain region at 24 h post fertilization. All infected cells express all heterologous proteins. Scale bar, 100 μm.