Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-01-22
2001-05-08
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S477000, C435S069100, C435S320100, C435S258300, C435S254100, C424S269100, C424S265100, C424S191100
Reexamination Certificate
active
06228649
ABSTRACT:
BACKGROUND OF THE INVENTION
Homologous gene replacement offers a powerful method for altering and testing gene function (Capecchi, M. (1989)
Science
244:1288-1292). In diploid organisms with a sexual cycle, a single heterozygous replacement is first obtained which is then rendered homozygous by sexual crossing. Although Leishmania are diploid at most loci, they appear to be predominantly or exclusively asexual in nature and in the laboratory (Tait, A. (1983)
Parasitology
86:29-57; Tibayrenc, M. et al. (1990)
Proc. Natl. Acad. Sci. USA
87:2414-2418; Panton, L. J. et al. (1991)
J. Protozoology
38:224-228). This situation is not unusual, as many diploid unicellular organisms lack a sexual cycle (Whelan, W. L. (1987)
CRC Critical Reviews
14:99-170) or possess one that is not readily manipulable.
Methods have been developed that can be utilized to obtain homozygous mutant lines from a heterozygous parent, in the absence of a manipulable sexual cycle. Parasexual crossing has been utilized in some organisms; however, parasex has not been demonstrated in organisms such as Leishmania. Another approach is the use of UV radiation or other agents to induce mitotic recombination following transformation, as shown in
Candida albicans
(Kelley, S. L. et al. (1988)
Science
241:1813-1815). The disadvantage is that mutagenic agents may create secondary mutations that cannot be removed by back-crossing.
Improved methods of homozygous gene replacement in diploid organisms which lack a sexual cycle are needed.
SUMMARY OF THE INVENTION
This invention pertains to methods of creating homozygous gene replacements resulting in complete loss of targeted gene function in asexual diploid organisms and to the mutant asexual diploid organisms carrying the homozygous gene replacements.
The method comprises targeting both alleles of the gene to be replaced with constructs employing two different, independent selectable markers. The first construct comprises DNA encoding a first selectable marker flanked by DNA complementary to flanking regions of one allele of the gene, or portion thereof, to be replaced and the second construct comprises DNA encoding the different selectable marker flanked by DNA complementary to flanking regions of the other allele of the gene, or portion thereof. The organism is transfected with the constructs which recombine and replace the targeted gene at each allele and transfected organisms are selected for expression of both selectable markers as indicative of homozygous replacement of the gene.
The method of this invention can be used in protozoans and in asexual, diploid yeast to enable functional genetic testing in these organisms. In addition, the method can be used to generate attenuated forms of parasitic organisms for use in live parasite vaccines or vaccine vehicles.
REFERENCES:
Johnston D.A. et al. “Genomics and the biology of parasites”. BioEssays vol. 21: pp. 131-147, Feb. 1999.*
Afrin, F. et al, “Isotype profiles of Leishmania donovani-infected Balb/c mice:” J. Parasitology, vol. 84, pp. 743-748, Aug. 1998.*
Kwiatkowski, D., “Development of a malaria vaccine”, The Lancet, vol. 350, pp. 1606-1701, Dec. 1997
Beverley Stephen M.
Cruz Angela K.
Guzo David
Lacourciere Karen A
President and Fellows of Harvard College
Testa Hurwitz & Thibeault LLP
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