Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-29
2002-03-12
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S252300, C435S253100
Reexamination Certificate
active
06355486
ABSTRACT:
BACKGROUND OF THE INVENTION
The World Health Organization estimates that one in three human beings is believed to be infected with
Mycobacterium tuberculosis
(Styblo, K.,
Reviews of Infectious Diseases
, Vol. II, Suppl. 2, March-April 1989; and Bloom and Murray,
Science
, 257:1055-1067 (1992)). Over the past decade, there has been a recent resurgence in the incidence of tuberculosis in developed countries that has coincided with the AIDS epidemic (Snider and Roper,
N. England J. Med
., 326:703-705 (1992)). Because of their impact as major human pathogens and as a result of their profound immunostimulatory properties, mycobacteria have long been intensively studied. In the early 1900s, an attenuated mycobacterium,
Mycobacterium
(
M.
)
bovis
Bacille Calmette-Guerin (
M. bovis
BCG or BCG), was isolated for use as a vaccine against tuberculosis (Calmette et al.,
Acad. Natl. Med
. (Paris), 91:787-796 (1924); reviewed in Collins, F. M.,
Bacterial Vaccines
(R. Germanier, ed.), Academic Press, pp. 373-418, 1984). Although the efficacy of this vaccine against tuberculosis varied considerably in different trials, and the reasons for its variable efficacy have yet to be resolved, BCG is among the most widely used human vaccines (Luelmo, F.,
Am. Rev. Respir. Dis
., 125:70-72 (1982); and Fine, P. E. M.,
Reviews of Infectious Diseases II
, Supp. 2:5353-5359 (1989)).
The recent application of molecular biological technology to the study of mycobacteria has led to the identification of many of the major antigens that are targets of the immune response to infection by mycobacteria (Kaufmann, S. H. E.,
Immunol. Today
, 11:129-136 (1990); Young, R. A.,
Ann. Rev. Immunol
., 8:401-420 (1990); Young et al., London:
Academic Press Ltd
., pp. 1-35, 1990; and Young et al.,
Mol. Microbiol
., 6:133-145 (1992)) and to an improved understanding of the molecular mechanisms involved in resistance to antimycobacterial antibiotics (Zhang et al.,
Nature
358:591-593 (1992); and Telenti et al,
Lancet
, 341:647-650 (1993). The development of tools that permit molecular genetic manipulation of mycobacteria has also allowed the construction of recombinant BCG vaccine vehicles (Snapper et al.,
Proc. Natl. Acad. Sci. USA
, 85:6987-6991 (1988); Husson et al.,
J. Bacteriol
., 172:519-524 (1990); Martin et al.,
B. Nature
, 345:739-743 (1990); Snapper et al.,
Mol. Microbiol
., 4:1911-1919 (1990); Aldovini and Young,
Nature
, 351:479-482 (1991); Jacobs et al.,
Methods Enzymol
., 204:537-555 (1991); Lee et al.,
Proc. Natl. Acad. Sci. USA
, 88:3111-3115 (1991); Stover et al.,
Nature
, 351:456-460 (1991); Winter et al.,
Gene
, 109:47-54 (1991); and Donnelly-Wu et al.,
Mol. Microbiol
., 7:407-417 (1993)). Genome mapping and sequencing projects are providing valuable information about the
M. tuberculosis
and
M. leprae
genomes that will facilitate further study of the biology of these pathogens (Young and Cole,
J. Bacteriol
., 175:1-6 (1993)).
Despite these advances, there are two serious limitations to our ability to manipulate these organisms genetically. First, very few mycobacterial genes that can be used as genetic markers have been isolated (Donnelly-Wu et al.,
Mol. Microbiol
., 7:407-417 (1993)). In addition, investigators have failed to obtain homologous recombination in slow growing mycobacteria, such as
M. tuberculosis
and
M. bovis
BCG (Kalpana et al.,
Proc. Natl. Acad. Sci. USA
, 88:5433-5447 (1991); and Young and Cole,
J. Bacteriol
., 175:1-6 (1993)), although homologous recombination has been accomplished in the fast growing
Mycobacterium smegmatis
(Husson et al.,
J. Bacteriol
., 172:519-524 (1990)).
SUMMARY OF THE INVENTION
Described herein is a method of transforming slow-growing mycobacteria, such as
M. bovis
BCG,
M. leprae, M. tuberculosis, M. avium, M. intracellulare
and
M. africanum
; a method of manipulating genomic DNA of slow-growing mycobacteria through homologous recombination; a method of producing homologously recombinant (HR) slow-growing mycobacteria in which heterologous DNA is integrated into the genomic DNA at a homologous locus; homologously recombinant (HR) slow-growing mycobacteria having heterologous DNA integrated into their genomic DNA at a homologous locus; and mycobacterial DNA useful as a genetic marker.
Applicants have succeeded in introducing heterologous DNA (i.e., transforming) into slow-growing mycobacteria through the use of electroporation in water (rather than in buffer). In the present method of transforming slow-growing mycobacteria, heterologous DNA (such as linear DNA or plasmid DNA) and slow-growing myco-bacteria (e.g.,
M. bovis
BCG,
M. leprae, M. tuberculosis, M. avium, M. intracellulare
and
M. africanum
) are combined and the resulting combination is subjected to electroporation at an appropriate potential and capacitance for sufficient time for the heterologous DNA to enter the slow growing mycobacteria, resulting in the production of transformed mycobacteria containing the heterologous DNA. In one embodiment, heterologous DNA and
M. bovis
BCG are combined and subjected to electroporation in water. In a particular embodiment, the
M. bovis
BCG-heterologous DNA combination is subjected to electroporation in water at settings of approximately 2.5 kV potential and approximately 25 &mgr;F capacitance. Optionally, prior to harvest, cells to be transformed are exposed to glycine (such as by adding 1-2% glycine to culture medium in which the slow-grow mycobacteria are growing) in order to enhance or improve transformation efficiencies. In one embodiment, 1.5% glycine is added to the culture medium 24 hours prior to harvesting of the cells, which are then combined with heterologous DNA to be introduced into the slow-growing mycobacteria. The resulting combination is subjected to electroporation, preferably in water, as described above.
In a further embodiment of the method of transforming slow growing mycobacteria, cultures of the cells are maintained in (continuously propagated in) mid-log growth, in order to increase the fraction of cells which are undergoing DNA synthesis (and which, thus, are competent to take up heterologous DNA). Cultures of cells maintained in log-phase growth are subjected to electroporation, preferably in water and, as a result, are transformed with the heterologous DNA. As described above, efficiency of transformation can be increased by exposing the slow-growing mycobacteria to glycine prior to electroporation. Thus, in this embodiment, slow-growing mycobacteria in log-phase growth are combined with heterologous DNA (e.g., plasmid DNA, linearized DNA) to be introduced into the slow-growing mycobacteria. The resulting combination is subjected to electroporation (preferably in water), under conditions (potential and capacitance settings and sufficient time) appropriate for transformation of the cells. Optionally, prior to electroporation, the log-phase cells are exposed to glycine (e.g., approximately 1-2% glycine added to culture medium) in order to enhance transformation efficiency.
Heterologous DNA introduced into slow-growing mycobacteria is DNA from any source other than the recipient mycobacterium. It can be homologous to DNA present in the recipient mycobacterial genomic DNA, nonhomologous or both. DNA which is homologous to mycobacterial genomic DNA is introduced into the genomic DNA by homologous recombination or integration. Alternatively, the heterologous DNA introduced by the present method can be nonhomologous and, thus, enter mycobacterial genomic DNA by random integration events or remain extrachromosomal (unintegrated) after it enters the mycobacterium. In addition, in one embodiment of the present method, nonhomologous DNA linked to or inserted within DNA homologous to genomic DNA of the recipient mycobacterium is introduced into genomic DNA of the recipient mycobacterium as a result of homologous recombination which occurs between genomic DNA and the homologous DNA to which the nonhomologous DNA is linked (or in which it is inserted). For example, as described herein, a mycobact
Aldovini Anna
Young Richard A.
Brusca John S.
Hamilton Brook Smith & Reynolds P.C.
Whitehead Institute for Biomedical Research
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