Chapter 3
Visualization of Bacteria-Mediated Gene Delivery Using
High-Resolution Electron and Confocal Microscopy
Andrew N. Osahor, Allan Wee Ren Ng, and Kumaran Narayanan
Abstract
Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally
performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to
the cell membrane. Standard microscopy techniques however fall short when finer details including
membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual
analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM)
to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the
vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging
the invasion complexes in high resolution can provide important insights into the behavior of bacterial
vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other
molecules.
Key words Cell membrane, Invasion, Electron microscopy, Confocal microscopy, Cell line,
Fluorescence
1 Introduction
Invasion of mammalian cells using invasive Escherichia coli (E. coli)
as a vector has continually been developed as a means to safely
deliver genes and therapeutic molecules [1]. Historically, successful
gene transfer to invaded cells is usually quantified via eukaryotic
expression of fluorescent proteins like Green Fluorescent Protein
(GFP) using standard fluorescence microscopy or Fluorescence
Activated Cell Sorting (FACS). These tools have proven sufficient
to determine transfection efficiencies during invasion experiments
[2–4], but do little to elucidate key interactions between the bacterial vectors and their target cells including vector–membrane interactions and the ability of the vector to internalize into the
cytoplasm.
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
29
Visualization of Bacteria-Mediated Gene Delivery Using
High-Resolution Electron and Confocal Microscopy
Andrew N. Osahor, Allan Wee Ren Ng, and Kumaran Narayanan
Abstract
Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally
performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to
the cell membrane. Standard microscopy techniques however fall short when finer details including
membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual
analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM)
to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the
vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging
the invasion complexes in high resolution can provide important insights into the behavior of bacterial
vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other
molecules.
Key words Cell membrane, Invasion, Electron microscopy, Confocal microscopy, Cell line,
Fluorescence
1 Introduction
Invasion of mammalian cells using invasive Escherichia coli (E. coli)
as a vector has continually been developed as a means to safely
deliver genes and therapeutic molecules [1]. Historically, successful
gene transfer to invaded cells is usually quantified via eukaryotic
expression of fluorescent proteins like Green Fluorescent Protein
(GFP) using standard fluorescence microscopy or Fluorescence
Activated Cell Sorting (FACS). These tools have proven sufficient
to determine transfection efficiencies during invasion experiments
[2–4], but do little to elucidate key interactions between the bacterial vectors and their target cells including vector–membrane interactions and the ability of the vector to internalize into the
cytoplasm.
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
29