US5773267A
Expired / Lapsed
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D29 shuttle phasmids and uses thereof
Assignee: Albert Einstein College of Medicine
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Filing: 1996-03-07
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Granted: 1998-06-30
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Expires: 2016-03-07
Abstract
Abstract The present invention provides a conditional shuttle phasmid constructed by inserting a cosmid into a non-essential region of the D29 mycobacteriophage which is capable of introducing DNA of interest into the chromosome of mycobacteria, especially M. tuberculosis complex organisms and other slow growing mycobacteria. The present invention provides a recombinant mycobacterium which expresses a DNA of interest incorporated into its chromosome by a conditional shuttle plasmid containing the DNA of interest. The present invention further provides a mycobacterial auxotrophic mutant and method of generating auxotrophic mutants. Finally, the present invention provides a method of inactivating a mycobacterial virulence gene.
Inventors
- 👤 William R. Jacobs
- 👤 Graham F. Hatfull
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 13:17
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C07K14/195; C07K14/35; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N7/00; C12Q; C12Q1/00; C12Q1/68; C12Q1/6897; C12N2795/00; C12N2795/00011; C12N2795/10011; C12N2795/10311; C12N2795/10322; C12N2795/10341; C12N2795/10343
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
17 words
A purified and isolated nucleic acid encoding D29 mycobacteriophage having the sequence of SEO ID NO: 1.
Claim 2
Dependent Claim
17 words
A purified and isolated nucleic acid encoding D29 mycobacteriophage having the sequence of SEO ID NO: 1.
Claim 3
Dependent Claim
24 words
A shuttle phasmid comprising a D29 mycobacteriophage which contains an E. coli bacteriophage lambda cosmid inserted into a non-essential region of said D29 mycobacteriophage.
Claim 4
Dependent Claim
24 words
A shuttle phasmid comprising a D29 mycobacteriophage which contains an E. coli bacteriophage lambda cosmid inserted into a non-essential region of said D29 mycobacteriophage.
Claim 5
Dependent Claim
13 words
The shuttle phasmid of claim 2 wherein said cosmid is excised by PacI.
Claim 6
Dependent Claim
15 words
The shuttle phasmid of claim 2, deposited under ATCC Accession No. 97468 and designated phAE70.
Claim 7
Dependent Claim
20 words
The shuttle phasmid of claim 2 further comprising a DNA of interest inserted into said E. coli bacteriophage lambda cosmid.
Claim 8
Dependent Claim
19 words
The shuttle phasmid of claim 3, that replicates at a temperature of 30° C., but not at 37° C.
Claim 9
Dependent Claim
14 words
The shuttle phasmid of claim 5, wherein said DNA of interest is a transposon.
Claim 10
Dependent Claim
31 words
The shuttle phasmid of claim 5, wherein said DNA of interest encodes a protein or polypeptide selected from the group consisting of: antigens, enzymes, lymphokines, immunopotentiators, selectable markers and reporter molecules.
Claim 11
Dependent Claim
20 words
A recombinant mycobacterium which expresses a DNA of interest introduced into its chromosome by the shuttle phasmid of claim 5.
Claim 12
Dependent Claim
34 words
An auxotrophic mutant produced by infecting a mycobacterium with the shuttle phasmid of claim 7, such that said transposon incorporates randomly or site-specifically into the genome of said mycobacterium thereby generating an auxotrophic mutant.
Claim 13
Dependent Claim
42 words
A method for producing a mutant mycobacterium comprising infecting a mycobacterium with the shuttle phasmid of claim 9 under conditions such that said transposon randomly or site-specifically inserts itself into the genome of said mycobacterium, thereby generating a mutation in said mycobacterium.
Claim 14
Dependent Claim
33 words
The recombinant mycobacterium of claim 9, wherein said DNA of interest encodes one or more proteins or polypeptides selected from the group consisting of: antigens, enzymes, lymphokines, immunopotentiators, selectable markers and reporter molecules.
Claim 15
Dependent Claim
14 words
The recombinant mycobacterium of claim 9, wherein said DNA of interest is a transposon.
Claim 16
Dependent Claim
16 words
The recombinant mycobacterium of claim 9, which is M. tuberculosis, M. bovis, BCG, or M. avium.
Claim 17
Dependent Claim
16 words
The auxotrophic mutant of claim 10 which is M. tuberculosis, M. bovis, BCG, or M. avium.
References Cited (Backward Citations)
No citation records indexed.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2001-06-29 | 2001-12-29 | 2002-06-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-06-29 | 2005-12-29 | 2006-06-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-06-29 | 2009-12-29 | 2010-06-30 | $7700.00 | Window Closed / Lapsed |