Gene Transfer Methodologies Research Articles

Introduction of foreign DNA into mycobacteria using a shuttle phasmid.

Mycobacteria are major pathogens of man and animals. There are approximately 10 million cases of tuberculosis world wide with an annual mortality of three million people. Leprosy, caused by Mycobacterium leprae, afflicts over ten million people, primarily in developing countries. M. tuberculosis and mycobacteria of the M. avium-intracellulare-scrofulaceum (MAIS) group are major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS). M. paratuberculosis is the cause of Jöhne's disease in cattle. Yet, BCG (Bacille Calmette-Guerin), an avirulent strain of M. bovis, is the most widely used human vaccine in the world, having been administered to about 2.5 X 10(9) people since 1948 (ref. 4). BCG was highly protective against tuberculosis in England, but has been found not to be effective in preventing pulmonary tuberculosis in adults in Southern India. We have initiated studies to develop the methodology for efficient gene transfer in mycobacteria. We have constructed recombinant shuttle phasmids which are chimaeras containing mycobacteriophage DNA into which an E. coli cosmid is inserted. They can replicate in E. coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera. These shuttle vectors permit for the first time the introduction of foreign DNA by infection into M. smegmatis and BCG. By introducing and ultimately expressing genes for protective antigens for a variety of pathogens, it may be possible to develop cultivatable mycobacteria into useful multivaccine vehicles.

Gene Transfer by Avian Retroviruses

Recent reports indicate that it is possible to transfer genetic material to the genomes of laboratory and domestic animals. This paper describes the progress in the development of a gene transfer methodology in the chicken using the avian retrovirus as a vector. Retroviruses are well suited as vectors for the transfer of exogenous genetic material, as during their life cycle they integrate into the host’s genome. We have chosen reticuloendotheliosis virus as a model system to establish the experimental conditions for integration of genetic material into the chicken genome. It was found that replication-competent virus can be transmitted to progeny by the inoculation of developing ovarian follicles in the reproductively active ovary. Further, it was found that a replication-defective vector carrying the herpes simplex thymidine kinase gene could be transmitted by a similar route.