A novel plasmid for delivering genes into mammalian cells with noninvasive food and commensal lactic acid bacteria - PubMed (original) (raw)

A novel plasmid for delivering genes into mammalian cells with noninvasive food and commensal lactic acid bacteria

Lin Tao et al. Plasmid. 2011 Jan.

Abstract

Using food and commensal lactic acid bacteria (LAB) as vehicles for DNA delivery into epithelial cells is a new strategy for vaccine delivery or gene therapy. However, present methods for DNA delivery with LAB have suffered low efficiency. Our goal was to develop a new system to deliver DNA into epithelial cells with high efficiency using food and commensal LAB. An Escherichia coli-LAB shuttle plasmid, pLKV1, for DNA delivery into eukaryotic cells was constructed. Two reporter plasmids with green and red fluorescent protein genes were also constructed to monitor the uptake of protein and DNA, respectively. Bacteria delivering these reporter plasmids into Caco-2 cells were monitored by fluorescence microscopy. Several methods that weaken the bacterial cell wall prior to co-culture with Caco-2 cells were evaluated for their role in the improvement of gene transfer efficiency. Treating Streptococcus gordonii with penicillin and lysozyme greatly increased its rate of gene delivery to mammalian cells compared to untreated control bacteria, while glycine pretreatment promoted the highest gene transfer rate for Lactococcus lactis. Uptake of green fluorescent bacteria by Caco-2 cells showed that the cell wall-weakening treatment promoted the internalization of the noninvasive bacteria into Caco-2 cells. In conclusion, we have developed a noninvasive system using LAB as a vehicle for vaccine delivery or gene therapy, and tested this system in vitro with Caco-2 cells.

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Figures

Fig. 1

Construction of pLKV1 and pLKV-Red2. The MCS in pLKV1 contains five unique restriction enzyme sites (from left to right): _Bam_HI, _Spe_I, _Eco_RI, _Pst_I, and _Not_I.

Fig. 2

Streptococcus gordonii V288 delivers the red fluorescent reporter plasmid pLKV-Red2 into Caco-2 cells with (A) and without (B) the penicillin and lysozyme treatment.

Fig. 3

Caco-2 cell uptake of GFPmut3*-expressing Lactococcus lactis NZ3900-pNZ8150-gfp with (A) and without (B) glycine treatment to weaken the bacterial cell wall. The phase contrast and fluorescent images were superimposed so that the unstained Caco-2 cells can be visualized.

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A novel plasmid for delivering genes into mammalian cells with noninvasive food and commensal lactic acid bacteria - PubMed (original) (raw)