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Last Updated: January 29, 2022

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Claims for Patent: 6,130,090

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Summary for Patent: 6,130,090
Title: Methods of performing gene trapping in bacterial and bacteriophage-derived artificial chromosomes and use thereof
Abstract:A method of efficiently sequencing multiple exons from complex genomic DNAs is disclosed. The methodology includes the use of bacterial and bacteriophage-derived artificial chromosomes (BBPACs) in novel gene trapping protocols. Targeted gene trapping by homologous recombination, and random gene trapping with the use of a transposon system are exemplified. Included in the invention are methods of preparing a gene map from BBPAC contigs, the resulting gene maps, methods of constructing a cDNA library from BBPAC contigs, and the resulting cDNA libraries.
Inventor(s): Heintz; Nathaniel (Pelham Manor, NY), Jiang; Weining (New York, NY), Yang; Xiangdong W. (New York, NY)
Assignee: The Rockefeller University (New York, NY)
Application Number:09/007,206
Patent Claims:1. A method of placing a eukaryotic promoter exon/intron unit (PEU) into a Bacterial or Bacteriophaze-Derived Artificial Chromosome (BBPAC) that contains a trappable eukaryotic gene comprising:

(a) introducing a conditional replication shuttle vector into a host cell containing the BBPAC under conditions in which the conditional replication shuttle vector can replicate and transform the host cell, wherein:

(i) the BBPAC contains a trappable eukaryotic gene, BBPAC vector DNA, and a second marker gene;

(ii) the conditional replication shuttle vector contains a RecA-like protein gene, a first marker gene which can be counter-selected against, and a recombination cassette, wherein the recombination cassette comprises a PEU flanked on both its 5' and 3' ends by nucleotide sequences that are homologous to BBPAC vector DNA; wherein the recombination cassette, the RecA-like protein gene, and the first marker gene are linked together on the conditional replication shuttle vector such that when the PEU integrates into the BBPAC, the RecA-like protein gene and the first marker gene remain linked together, but neither the RecA-like protein gene nor the first marker gene remain linked to the integrated PEU;

(iii) the PEU comprises a third marker gene, a eukaryotic promoter, at least one 5' vector-derived exon, and an intron or fragment thereof; wherein the 5' vector-derived exon is adjacent to the intron or fragment thereof and is operatively downstream from the eukaryotic promoter; wherein when the trappable eukaryotic gene comprises an exon with a 3' splice acceptor, the PEU can integrate into the BBPAC placing the exon of the trappable eukaryotic gene operatively downstream of the PEU; and

(iv) wherein neither the host cell nor the BBPAC independently or in conjunction can support homologous recombination, without the conditional replication shuttle vector;

(b) growing the host cell under conditions in which the conditional replication shuttle vector can replicate, the RecA-like gene can be expressed, and in which a cell that contains the first and second marker genes is selected for; and wherein a first homologous recombination event occurs between the recombination cassette and the BBPAC to form a co-integrate;

(c) growing the cell selected for in step (b) under conditions in which the conditional replication shuttle vector cannot replicate and in which a cell that contains the first and second marker proteins is selected for, whereby a cell containing the co-integrate between the recombination cassette and the BBPAC is selected for; and

(d) growing the cell selected for in step (c) under conditions in which the conditional replication shuttle vector cannot replicate and in which a cell that contains the second marker gene is selected for; wherein a second homologous recombination event occurs between the conditional replication shuttle vector and the BBPAC; and wherein the PEU is placed into the BBPAC.

2. The method of claim 1 wherein the PEU does not contain an exon encoding a 3' polyadenylation sequence.

3. A method of isolating a cell that contains a BBPAC having a eukaryotic promoter exon/intron unit (PEU) and a trappable eukaryotic gene comprising growing the cell selected for in step (d) of claim 1 under conditions in which a cell that contains the second and third marker genes is selected for, while a cell that contains the first marker gene is selected against; wherein a cell containing a BBPAC having a eukaryotic promoter exon/intron unit (PEU) is isolated.

4. The method of claim 3 wherein the first marker gene is a tetracycline resistance gene that can be counter-selected against by growing the cell in the presence of fusaric acid.

5. The method of claim 1 wherein the PEU is a bi-directional eukaryotic promoter-exon/intron unit (BPEU).

6. The method of claim 1 wherein the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) having a temperature-sensitive origin of replication, wherein the TSSV replicates at a permissive temperature, but does not replicate at a non-permissive temperature.

7. The method of claim 1 wherein the BBPAC is a BAC.

8. The method of claim 1 wherein the PEU comprises two 5' vector-derived exons and one intron.

9. The method of claim 8 wherein the two 5' vector-derived exons consist of the first exon of beta-globin and a fusion exon containing the second exon of beta-globin fused to the HIV-tat exon; the intron is the HIV-tat intron; and wherein the fusion exon is adjacent to the HIV-tat intron.

10. A method of transcribing a trappable eukaryotic gene contained in a BBPAC in a eukaryotic cell comprising:

(a) isolating the BBPAC containing the trappable eukaryotic gene operably downstream of the PEU from the isolated cell of claim 3;

(b) transfecting the isolated BBPAC into a eukaryotic cell; and

(c) culturing the eukaryotic cell; wherein when the PEU is operatively upstream to an exon of the trappable eukaryotic gene, the eukaryotic promoter of the PEU facilitates the transcription of the exon of the trappable eukaryotic gene into an MRNA; wherein the trappable eukaryotic gene is transcribed.

11. A method of determining the nucleotide sequence of a trappable eukaryotic gene contained in a BBPAC comprising:

(a) preparing a cognate cDNA of the mRNA of claim 10 as a template; and

(b) determining the nucleotide sequence of the cognate cDNA; wherein the nucleotide sequence of the trappable eukaryotic gene contained in the BBPAC is determined.

12. The method of claim 11 wherein preparing the cognate cDNA is performed by PCR.

13. A method of placing a eukaryotic promoter exon/intron unit (PEU) into a BBPAC that contains a trappable eukaryotic gene comprising:

(a) introducing a shuttle vector into a host cell containing the BBPAC under conditions in which the shuttle vector can replicate and transform the host cell wherein:

(i) the BBPAC contains a trappable eukaryotic gene, BBPAC vector DNA, and a second marker gene;

(ii) the shuttle vector comprises a first marker gene which can be counter-selected against, the PEU, a mini-transposon containing a pair of inverted transposon ends, a nucleic acid encoding transposase, and an inducible promoter; wherein the expression of transposase is under the control of the inducible promoter; wherein the PEU is positioned in between the pair of inverted transposon ends; and the nucleic acid encoding transposase, the inducible promoter, and the first marker gene are positioned outside of the pair of inverted transposon ends; and

(iii) the PEU comprises a third marker gene, a eukaryotic promoter, at least one 5' vector-derived exon, and an intron or fragment thereof; wherein a 5' vector-derived exon is adjacent to an intron or fragment thereof and is operatively downstream from the eukaryotic promoter; wherein when the trappable eukaryotic gene comprises an exon with a 3' splice acceptor, the PEU can integrate into the BBPAC and place the exon of the trappable eukaryotic gene operatively downstream of the PEU;

(b) growing the host cell under conditions in which the shuttle vector can replicate, and in which a cell that contains the first and second marker gene are selected for; and

(c) inducing the inducible promoter of the cell selected for in step (b), wherein transposase is expressed; and wherein the PEU is placed into the BBPAC.

14. The method of claim 13 wherein the PEU does not contain an exon encoding a 3' polyadenylation sequence.

15. The method of claim 13 wherein the shuttle vector is a conditional replication shuttle vector.

16. A method of isolating a cell that contains a BBPAC having a eukaryotic promoter exon/intron unit (PEU) and a trappable eukaryotic gene comprising growing the cell selected for in step (c) of claim 15 under conditions in which the conditional replication shuttle vector cannot replicate, and in which a cell that contains the second and third marker genes are selected for, while a cell that contains the first marker gene is selected against; wherein a cell containing a BBPAC having a eukaryotic promoter exon/intron unit (PEU) is isolated.

17. The method of claim 16 wherein the first marker gene is a tetracycline resistance gene that can be counter-selected against by growing the cell in the presence of fusaric acid.

18. The method of claim 15 wherein the PEU is a bidirectional eukaryotic promoter-exon/intron unit (BPEU).

19. The method of claim 15 wherein the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) having a temperature-sensitive origin of replication, wherein the TSSV replicates at a permissive temperature, but does not replicate at a non-permissive temperature.

20. The method of claim 15 wherein the BBPAC is a BAC.

21. The method of claim 15 wherein the inducible promoter is the .beta.-galactosidase promoter; wherein the host cell expresses lacI.sup.q ; and wherein said inducing of the inducible promoter comprises contacting the bacterial host cell with IPTG.

22. The method of claim 15 wherein the amount of IPTG used to contact the bacterial host is controlled so that the BBPAC receives only a single PEU.

23. The method of claim 15 wherein the inducible promoter is the .beta.-galactosidase promoter; wherein the TSSV encodes lacI.sup.q ; and wherein said inducing of the inducible promoter comprises contacting the bacterial host cell with IPTG.

24. The method of claim 23 wherein the amount of IPTG used to contact the bacterial host is controlled so that the BBPAC receives only a single PEU.

25. A method of purifying a BBPAC having a eukaryotic promoter exon/intron unit (PEU) comprising

(a) performing alkaline lysis of the isolated cell obtained from claim 16, wherein the BBPAC DNA is isolated;

(b) electroporating the isolated BBPAC DNA into competent bacterial cells;

(c) growing the competent bacterial cells under conditions in which the conditional replication shuttle vector cannot replicate and in which cells that contain the second and third marker genes are selected for; and

(d) performing alkaline lysis of the bacterial cells selected for in step (c); wherein the BBPAC DNA is purified.

26. A method of transcribing a trappable eukaryotic gene contained in a BBPAC in a eukaryotic cell comprising:

(a) transfecting the purified BBPAC from step (d) of claim 25 into a eukaryotic cell; and

(b) culturing the eukaryotic cell; wherein when the BBPAC contains a eukaryotic promoter exon/intron unit (PEU) operatively upstream to an exon of the trappable eukaryotic gene, the eukaryotic promoter facilitates the transcription of the exon of the trappable eukaryotic gene into an mRNA; wherein the trappable eukaryotic gene is transcribed.

27. A method of determining the nucleotide sequence of a trappable eukaryotic gene contained in a BBPAC comprising:

(a) preparing a cognate cDNA of the MRNA of claim 26 as a template; and

(b) determining the nucleotide sequence of the cognate cDNA; wherein the nucleotide sequence of a trappable eukaryotic gene contained in the BBPAC is determined.

28. The method of claim 27 wherein preparing the cognate cDNA is performed by PCR.

29. The method of claim 15 further comprising mapping the insertion site of the PEU.

30. The method of claim 29 wherein the mapping is performed by a procedure selected from the group consisting of pulse field gel electrophoresis and Southern blots.

Details for Patent 6,130,090

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 1986-06-04 ⤷  Try it Free 2017-06-23
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try it Free 2017-06-23
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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