In vitro production of the obligate intracellular bacterium, Wolbachia pipientis, is essential to its manipulation as a genetic tool to spread transgenes within vector populations. We have adapted the Wolbachia-infected Aa23 Aedes albopictus mosquito cell line to Eagle's minimal medium, supplemented with nonessential amino acids, glutamine, and 20% fetal bovine serum. When plated at low densities, Aa23E cells grew as patchy monolayers, comprised of non-contiguous clusters of cells that gave rise to solid clumps of tightly adherent cells. Multicellular clumps eventually detached from the substrate and floated freely in the medium. Removal of Wolbachia by treatment with tetracycline did not alter the cytological properties of the host cells, which had a population doubling time of 4-5 d. The presence of Wolbachia was monitored by Giemsa staining of cytological preparations, polymerase chain reaction (PCR) amplification of Wolbachia 16S ribosomal DNA, and by simultaneous PCR amplification of ribosomal protein genes from Wolbachia and mosquito host cell genomes. Wolbachia morphology was pleomorphic, and Wolbachia DNA persisted in the culture medium for several weeks after degradation of PCR-amplifiable host cell DNA.
|Original language||English (US)|
|Number of pages||8|
|Journal||In Vitro Cellular and Developmental Biology - Animal|
|State||Published - Jun 2008|
Bibliographical noteFunding Information:
Acknowledgments This work was supported by NIH grant AI070913 and by the University of Minnesota Agricultural Experiment Station, St. Paul, MN. I thank Anna Gerenday for technical assistance and Drs. TJ Kurtti, UG Munderloh and A Gerenday for helpful discussions.
Copyright 2008 Elsevier B.V., All rights reserved.
- Aedes albopictus
- Giemsa stain
- Intracellular bacteria
- Mosquito cell line
- Polymerase chain reaction (PCR)