Processing

Please wait...

Settings

Settings

Goto Application

1. WO2000017342 - INTRON-MEDIATED RECOMBINANT TECHNIQUES AND REAGENTS

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

Claims
1. A method for producing recombinant nucleic acids in vivo, the method comprising:
providing a first DNA construct, including:
a first promoter;
a first exon;
a first infron component; and
a means of terminating transcription;
providing a second DNA construct, including:
a second promoter;
a second intron component selected to be capable of directing trans-splicing when contacted with the first intron component;
a second exon; and
a means of terminating transcription;
introducing each of the first and second DNA constructs into a cell so that both the first and second promoters are expressed and two transcripts are produced in the cell;
a first transcript including the first exon and the first intron component; and
a second transcript including the second intron component and the second exon; and
allowing the first and second transcripts to trans-splice with one another so that a product transcript including each of the first and second exons, but lacking the first and second infron components, is produced.

2. The method of claim 1, further including a step of:
providing a third DNA construct, including:
a third promoter; and
a third exon flanked by intron components,
and wherein the method of introducing includes introducing the third DNA construct into the cell so that a third franscript is produced including the third exon flanked by infron components; and the step of allowing comprises allowing all three transcripts to trans-splice.

3. The method of claim 2 wherein the step of providing a third construct comprises providing a plurality of third constructs.

4. The method of any one of claims 1-3 wherein at least one of the DNA constructs includes an origin of replication.

5. The method of any one of claims 1-3 wherein at least one of the DNA constructs does not include an origin of replication.

6. The method of any one of claims 1-3 wherein the step of introducing comprises introducing at least one DNA construct into the cell so that a least a portion thereof integrates into a DNA already present in the cell and the encoded transcript is expressed from the integrated construct.

7. The method of any one of claims 1-3 wherein at least one of the promoters is inducible.

8. A streamlined method of trans-splicing, comprising steps of:
(i) providing a first RNA exon linked to a first intron component;
(ii) providing a second RNA exon linked to a second intron component that is compatible with the first intron component;
(iii) allowing the first and second exons to be linked by trans-splicing; (iv) copying the trans-splicing product into DNA; and
(v) introducing the DNA product into a vector,
wherein no gel-purification step is performed between steps (iii) and (iv), or between steps (iv) and (v).

9. A kit comprising:
a first vector comprising:
a first promoter;

a first infron component;
a first exon cloning site; and
a first means of terminating transcription; and
a second vector comprising:
a second promoter;
a second exon cloning site;
a second intron component, compatible with the first infron component; and
a second means of terminating transcription.

10. The kit of claim 9, further including one or more of:
transcription reagents;
splicing buffer;
reverse transcription reagents;
polymerase chain reaction reagents;
DNA cloning reagents; and
DNA sequencing reagents.

11. The kit of either of claims 9 or 10, further including:
a third vector comprising:
a third promoter;
a third exon cloning site flanked by intron components; and
a third means of terminating transcription.

12. The kit of claim 11 wherein at least one pair of intron components is incompatible for trans-splicing.

13. Inventive vectors.