Customizable BAC-based DNA markers for pulsed-field gel electrophoresis.

DNA markers are used as a size reference and sample loading control during gel electrophoresis. Most markers are designed for conventional gel electrophoresis to separate DNA smaller than 20 kb. For larger molecules, pulsed-field gel electrophoresis (PFGE) marker is required. Limited PFGE markers are available because large DNA are prone to nicking and degradation, causing smeary bands. Here, we developed a robust marker based on bacterial artificial chromosomes (BACs) with bands up to 184 kb. This marker could consistently confer intense and distinct bands for accurate gel analysis in molecular biology studies, laboratory validations or clinical diagnosis.

Large BACs transfect more efficiently in circular topology.

The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.

Improved DNA Delivery Efficiency of Bacterial Vectors by Co-Delivery with Exogenous Lipid and Antimicrobial Reagents.

Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting ß1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.

Visualization of Bacteria-Mediated Gene Delivery Using High-Resolution Electron and Confocal Microscopy.

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.

Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis.

Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.

Short homologies efficiently generate detectable homologous recombination events.

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.