J Biotechnol. 2013 Jul 20;166(4):182-6.

Evaluation of cre recombinase delivery in mammalian cells using baculovirus infection.

Erbs E, Pradhan AA, Matifas A, Kieffer BL, Massotte D.

Department of Neurobiology and Translational Medicine, IGBMC, Illkirch, France. erbs@igbmc.fr

 

ABSTRACT:

In vivo conditional knock-out of a protein is a method of choice to decipher its biological function. It can be achieved by encoding the cre-recombinase on a recombinant virus to exert spatio-temporal control of its expression and enzymatic activity and, subsequently, of the target gene deletion. Recombinant baculoviruses have been successfully used to express a wide range of proteins in insect cells. More recently, their potential to infect mammalian cells has been addressed but, so far, their ability to yield a conditional knock-out as a result of efficient in vivo cre-recombinase gene delivery has not been examined. Cre-recombinase fused to the green fluorescent protein was cloned under the control of the CAG promoter in a recombinant Autographa californica baculovirus expressing the vesicular stomatitis virus envelope G protein for increased mammalian cell infection. Gene delivery was evaluated in vitro in mammalian cells, neuroblastoma and mouse primary neuronal cultures as well as in vivo in the mouse brain. Infection with adeno-associated viruses encoding the cre-recombinase fused to the green fluorescent protein was performed as a positive control. Our results indicate that baculovirus infection leads to functional cre-recombinase expression in non-neuronal and neuroblastoma cell lines but not in mouse primary neuronal cultures or brain. Copyright © 2013 Elsevier B.V.

KEYWORDS: Adeno associated virus, Baculovirus, Cre recombinase, Mammalian cell, Mouse brain

PMID: 23732834

 

SUPPLEMENT:

Recombinant baculoviruses, mainly Autographa californica multiply embedded nuclear polyhedrosis viruses (ACMNPV), have been widely used for heterologous expression of soluble as well as membrane proteins with various intracellular and extracellular functions. These viruses naturally infect insects but can also infect mammalian cells though they cannot replicate in the latter cell types [1]. Baculoviruses are therefore considered as safe viral vectors for human gene delivery [2-3].

Various strategies were developed to increase infection of mammalian cells by baculoviruses. Among them, expression of the glycoprotein of the vesicular stomatitis virus (VSVG) in the viral envelope was previously described as significantly enhancing adhesion to the cell, which led to improved expression of the luciferase reporter gene in several cell lines of mammalian origin [4]. Few attempts have however been published regarding baculovirus-dependent gene delivery in neurons. Moreover, they only explored the expression of three cytoplasmic reporter genes: the green fluorescent protein eGFP [4-6], luciferase [7-10], and β-galactosidase [11].

In this context, we evaluated whether recombinant baculoviruses expressing the VSVG protein [4] were suitable to successfully drive the expression of functional nuclear proteins in mammalian cells including neurons. We chose the cre-recombinase as a model because this enzyme is widely used to create conditional knock-out mice. Indeed, its enzymatic activity enables spatio-temporal control of the expression of a transgene flanked by two loxP sites.

The cre recombinase was fused to its N-terminus to the fluorescent protein eGFP for easy detection and expression was driven by the ubiquitous CAG promoter. Successful nuclear expression of a functional cre-recombinase and efficient excision of a target sequence were observed following infection of mammalian (COS and HEK 293) and neuroblastoma (neuro2A) cell lines (Figure 1).

Figure-12

Successful in vitro expression of the luciferase reporter gene in mouse hippocampal primary neurons demonstrated the ability of the recombinant baculoviruses to infect primary neurons and to drive expression of cytoplasmic reporter genes as previously described [4, 5, 7, 8, 11]. We were however unable to detect cre recombinase enzymatic activity in primary neurons or in the mouse brain. Interestingly, the eGFP fluorescence was mainly detected in the cytoplasm instead of the nucleus (Figure 2) though the fluorescence associated with the eGFP-cre fusion was present in the nucleus of neurons upon infection with adeno-associated viruses (AAV) (Figure 2). Further investigation is now needed to establish whether the non-dividing nature of neurons and/or deficient nuclear import contribute to the abnormal localization observed following baculovirus infection.

figure2
Figure 2: eGFP-Cre expression (in green) in mouse primary neurons is detected in the nucleus (labeled with DAPI) following infection with adeno-associated viruses (left panel) or in the nucleus and cytoplasm following infection with recombinant VSVG-modified baculoviruses (right panel).

 

Importantly, our study also established that VSVG-modified recombinant baculoviruses exhibit in vitro tropism towards neurons comparable to serotypes 1 or 2 AAVs. Also, we showed that the volume of infection remained limited in vivo, which is consistent with infection by a virus that cannot replicate in mammalian cells [1] and emphasizes the potential of recombinant baculoviruses as safe viral vectors for human gene delivery [2].

Altogether, our work demonstrates that recombinant VSVG-modified baculoviruses are suitable for efficient delivery of genes encoding proteins with nuclear activity in dividing mammalian cells and therefore represent a highly attractive option for human therapy.

Contact address: d.massotte@unistra.fr

 

References

1. Hofmann, C., et al., Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S A, 1995. 92(22): p. 10099-103.

2. Li, S.F., et al., Genetic modification of baculovirus expression vectors. Virol Sin, 2012. 27(2): p. 71-82.

3. Airenne, K.J., et al., Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther, 2013. 21(4): p. 739-49.

4. Tani, H., et al., In vitro and in vivo gene delivery by recombinant baculoviruses. J Virol, 2003. 77(18): p. 9799-808.

5. Sarkis, C., et al., Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc Natl Acad Sci U S A, 2000. 97(26): p. 14638-43.

6. Wang, J., et al., Efficient transduction of spiral ganglion neurons in vitro by baculovirus vectors. Neuroreport, 2007. 18(13): p. 1329-33.

7. Wang, C.Y. and S. Wang, Adeno-associated virus inverted terminal repeats improve neuronal transgene expression mediated by baculoviral vectors in rat brain. Hum Gene Ther, 2005. 16(10): p. 1219-26.

8. Wang, X., et al., Gene transfer to dorsal root ganglia by intrathecal injection: effects on regeneration of peripheral nerves. Mol Ther, 2005. 12(2): p. 314-20.

9. Li, Y., Y. Yang, and S. Wang, Neuronal gene transfer by baculovirus-derived vectors accommodating a neurone-specific promoter. Exp Physiol, 2005. 90(1): p. 39-44. 10. Li, Y., et al., Axonal transport of recombinant baculovirus vectors. Mol Ther, 2004. 10(6): p. 1121-9.

11. Lehtolainen, P., et al., Baculoviruses exhibit restricted cell type specificity in rat brain: a comparison of baculovirus- and adenovirus-mediated intracerebral gene transfer in vivo. Gene Ther, 2002. 9(24): p. 1693-9.

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