to allow successful adherence of the vector to the cell membrane
and subsequent internalization into the cytoplasm [16]. Using this
method, the invasive bacteria are simply mixed and incubated with
lipids or antimicrobial reagents which are then co-delivered during
invasion. Complexing these reagents with invasive E. coli prior to
invasion of host cells provides vastly improved membrane adherence and internalization capabilities to the vectors with minimal
toxicity to the targeted cell line (Figs. 2 and 3).
Using this method, we have recorded up to eight-fold increase
in reporter expression in invaded cell populations when cationic
lipids, and antimicrobial reagents are complexed with E. coli cells.
Subsequently, we have observed that these reagents improve the
vectors ability to deliver DNA to cells by greatly improving adherence to the cell membrane while simultaneously attenuating the
vector, rendering them prone to easily release of their genetic cargo
once inside the cell.
In this chapter, we describe methods to co-deliver invasive
E. coli with selected lipids and antimicrobial reagents to enhance
the gene delivery efficiency of this vector. This method is likely to
be applicable for other bacterial vectors including Listeria and
Salmonella, or for cell lines that are inherently difficult to target
using bacterial vectors.
2 Materials
Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Sterilize all reagents and culture vessels to avoid contamination. Diligently follow all waste disposal
regulations when disposing waste materials.
2.1 Invasive E. coli
Vector Preparation
1. Bacterial growth media: Brain Heart Infusion (BHI) media.
Prepare according to manufacturer’s instructions (see Note 1)
and transfer to a 250 mL conical flask. Autoclave to sterilize.
Store at room temperature.
2. Invasive E. coli vector: E. coli DH10B (pGB2Ωinv-hly,
pEGFPN2) [17] (see Note 2).
3. Noninvasive control vector: E. coli DH10B (pEGFPN2) [17].
4. Cell lines: HEK 293 (ATCC #: CRL-1573), HeLa (ATCC #:
CCL-2), HT1080 (ATCC #: CCL-121) (see Note 3).
5. Fluorescence-activated cell sorting: FACSCalibur flow cytometer (Beckton Dickinson) running CellQuest Pro V 6.0
software.
Enhancing Bactofection with Exogenous Reagents
17
and subsequent internalization into the cytoplasm [16]. Using this
method, the invasive bacteria are simply mixed and incubated with
lipids or antimicrobial reagents which are then co-delivered during
invasion. Complexing these reagents with invasive E. coli prior to
invasion of host cells provides vastly improved membrane adherence and internalization capabilities to the vectors with minimal
toxicity to the targeted cell line (Figs. 2 and 3).
Using this method, we have recorded up to eight-fold increase
in reporter expression in invaded cell populations when cationic
lipids, and antimicrobial reagents are complexed with E. coli cells.
Subsequently, we have observed that these reagents improve the
vectors ability to deliver DNA to cells by greatly improving adherence to the cell membrane while simultaneously attenuating the
vector, rendering them prone to easily release of their genetic cargo
once inside the cell.
In this chapter, we describe methods to co-deliver invasive
E. coli with selected lipids and antimicrobial reagents to enhance
the gene delivery efficiency of this vector. This method is likely to
be applicable for other bacterial vectors including Listeria and
Salmonella, or for cell lines that are inherently difficult to target
using bacterial vectors.
2 Materials
Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Sterilize all reagents and culture vessels to avoid contamination. Diligently follow all waste disposal
regulations when disposing waste materials.
2.1 Invasive E. coli
Vector Preparation
1. Bacterial growth media: Brain Heart Infusion (BHI) media.
Prepare according to manufacturer’s instructions (see Note 1)
and transfer to a 250 mL conical flask. Autoclave to sterilize.
Store at room temperature.
2. Invasive E. coli vector: E. coli DH10B (pGB2Ωinv-hly,
pEGFPN2) [17] (see Note 2).
3. Noninvasive control vector: E. coli DH10B (pEGFPN2) [17].
4. Cell lines: HEK 293 (ATCC #: CRL-1573), HeLa (ATCC #:
CCL-2), HT1080 (ATCC #: CCL-121) (see Note 3).
5. Fluorescence-activated cell sorting: FACSCalibur flow cytometer (Beckton Dickinson) running CellQuest Pro V 6.0
software.
Enhancing Bactofection with Exogenous Reagents
17