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US 2003/0224481 A1Published application
In vivo gene transfer
AssigneeBaylor College of Medicine
InventorsStephen J. Elledge, Mamie Z. Li
Life of this patent
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Abstract
The present invention is directed to an in vivo method of transferring DNA from a donor cell to a recipient cell. In a specific embodiment, the method comprises a highly efficient process of bacterial mating. In a preferred embodiment, selection for a recombinant plasmid against a parent host plasmid and donor plasmid is based on recircularization of the host plasmid by its recombination with a gene of interest such that it is now no longer cleaved by a restriction enzyme expressed in the recipient cell.
Claims70
A method of DNA transfer, comprising:
- introducing into an E. coli bacteria a nucleic acid molecule comprising:
- a polynucleotide of interest;
- a first homology region;
- and
- a second homology region,
- wherein the bacteria comprises a vector comprising:
- a first homology region;
- and
- a second homology region;
- and
- homologously recombining said polynucleotide of interest into said vector at said homology regions, wherein the nucleic acid molecule comprises a double strand break, the vector comprises a double strand break, or both the nucleic acid molecule and the vector comprise double strand breaks.
8 more elements
A method of DNA transfer in vivo, comprising the steps of:
- conjugating a first bacteria having a donor vector comprising a polynucleotide of interest with a second bacteria having a recipient vector, wherein the conjugation results in transfer of the donor vector to the second bacteria;
- and
- inserting the polynucleotide of interest from the donor vector into the recipient vector by homologous recombination.
A method of DNA transfer, comprising the steps of:
- providing a vector transfer means;
- providing a first bacteria having a first vector, wherein the first vector comprises:
- 1) a cassette comprising in an operably linked manner and in a 5′ to 3′ direction:
- a) a first rare restriction enzyme cutting site;
- b) a first homology region;
- c) a polynucleotide of interest;
- d) a second homology region;
- and
- e) a second rare restriction enzyme cutting site;
- 2) a first origin of replication;
- and
- 3) a first selection marker;
- providing a second bacteria capable of receiving said first vector, said bacteria having
- 1) a conditionally regulatable polynucleotide encoding said rare restriction enzyme;
- 2) a second vector, wherein the second vector comprises
- a) a cassette comprising in an operably linked manner and in a 5′ to 3′ direction:
- 1) a first homology region;
- 2) a first rare restriction enzyme cutting site;
- 3) a second rare restriction enzyme cutting site;
- and
- 4) a second homology region;
- b) a second selection marker;
- c) a second origin of replication;
- and
- 3) recombination means;
- and
- contacting said first bacteria with said second bacteria under conditions wherein said first vector is transferred to said second bacteria by bacterial conjugation and said polynucleotide of interest is inserted by homologous recombination into said second vector.
24 more elements
A method of DNA transfer in vivo, comprising the steps of:
- providing a first bacteria having the genotype F′ pir-116;
- providing a second bacteria having the genotype umuC::PBAD-I-SceI;
- contacting said first bacteria with said second bacteria under conditions wherein a first vector having a polynucleotide of interest is transferred to said second bacteria by bacterial conjugation and said polynucleotide of interest is inserted by homologous recombination into said second vector.
A method of cloning a polynucleotide of interest into a vector, comprising the steps of:
- providing a DNA molecule having in a 5′ to 3′ direction:
- a first homology region;
- the polynucleotide of interest;
- and
- a second homology region;
- and
- transferring said DNA molecule into a bacteria having a recipient vector comprising:
- an origin of replication;
- a selection marker;
- and
- an expression casette comprising in a 5′ to 3′ direction:
- a first homology region;
- at least one conditionally lethal gene;
- and
- a second homology region;
- generating a double strand break in said vector, wherein the break results in liberation of the conditionally lethal gene;
- homologously recombining the polynucleotide of interest into the recipient vector through the first and second homology regions;
- and
- counterselecting with said conditionally lethal gene product against bacteria wherein said homologous recombination did not occur.
16 more elements
A system for DNA transfer, comprising components selected from the group consisting of:
- a first vector, wherein the first vector comprises:
- a) a cassette comprising in an operably linked manner and in a 5′ to 3′ direction:
- 1) a first rare restriction enzyme cutting site;
- 2) a first homology region;
- 3) a second homology region;
- 4) a second rare restriction enzyme cutting site;
- b) a first origin of replication;
- and
- c) a first selection marker;
- and
- a second vector, wherein the second vector comprises
- a) a cassette comprising in an operably linked manner and in a 5′ to 3′ direction:
- 1) a first homology region;
- 2) at least one rare restriction enzyme cutting site;
- 3) a second homology region;
- b) a second selection marker;
- c) a second origin of replication;
- and
- a combination thereof.
16 more elements
A system for DNA transfer, comprising components selected from the group consisting of:
- a vector transfer means;
- a first bacteria having
- 1) a first vector, wherein the first vector comprises:
- a) a cassette comprising in an operably linked manner and in a 5′ to 3′ direction:
- 1) a first rare restriction enzyme cutting site;
- 2) a first homology region;
- 3) a second homology region;
- 4) a second rare restriction enzyme cutting site;
- b) a first origin of replication;
- and
- c) a first selection marker;
- a second bacteria having
- 1) a conditionally regulatable polynucleotide encoding said rare restriction enzyme;
- 2) a second vector, wherein the second vector comprises
- a) a cassette comprising in an operably linked manner and in a 5′ to 3′ direction:
- 1) a first homology region;
- 2) at least one rare restriction enzyme cutting site;
- 3) a second homology region;
- b) a second selection marker;
- c) a second origin of replication;
- and
- 3) recombination means.
19 more elements
Dependencies are inferred from the claim text.