A high-throughput comparison of recombinant gene expression parameters for E. coli-mediated gene transfer to P388D1 macrophage cells.
Escherichia coli strain BL21(DE3) was tested as a delivery vector for gene transfer to a murine P388D1 macrophage cell line using a 96-well high-throughput assay. Five recombinant strains of E. coli were compared to identify the effect recombinant listeriolysin O (LLO) and associated gene expression parameters had on final delivery of a luciferase reporter gene. Listeriolysin O, native to Listeria monocytogenes and used here in an effort to improve final gene delivery, was expressed from plasmid and chromosomal locations under the control of constitutive Tet or inducible T7 promoters. The E. coli vectors delivered the luciferase reporter gene to the P388D1 line with success assessed by recording luciferase luminescence activity within the macrophage cells. The assay allowed rapid analysis and evaluation of each E. coli strain tested with strain BL21(DE3) harboring a chromosomal copy of the T7-driven LLO gene showing the greatest relative measure of gene delivery. Strains were separately assayed for LLO activity and exhibited a trend of maximum gene delivery between the lowest and highest recorded LLO activities.
Parsa
Saba
S
Department of Chemical & Biological Engineering, Tufts University, Medford, MA 02155, United States.
Wang
Yong
Y
Rines
Katie
K
Pfeifer
Blaine A
BA
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism*
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D008156:Luciferases; C038705:hlyA protein, Listeria monocytogenes