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Last Updated: December 6, 2021

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Claims for Patent: 6,156,574

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Summary for Patent: 6,156,574
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:09/102,488
Patent Claims:1. A method of placing a eukaryotic promoter exon/intron unit (PEU) into a Bacterial or Bacteriophage-Derived Artificial Chromosome (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 the trappable eukaryotic gene, BBPAC vector DNA, and a second marker gene;

(ii) the shuttle vector comprises the PEU and a first marker gene which can be counter-selected against; wherein the PEU can be transferred from the shuttle vector to the BBPAC while the first marker gene remains with the shuttle vector; 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 the intron or fragment thereof, and is operatively downstream from the eukaryotic promoter; and 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;

(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 is selected for; and

(c) transferring the PEU from the shuttle vector to the BBPAC of the cell selected for in step (b), wherein the first marker gene remains with the shuttle vector, 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. The method of claim 1 wherein the shuttle vector is a conditional replication shuttle vector.

4. 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 obtained following the PEU transferring step of claim 3 (c) under conditions in which the conditional replication shuttle vector cannot replicate; wherein 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; and wherein a cell containing a BBPAC having a eukaryotic promoter exon/intron unit (PEU) is isolated.

5. The method of claim 3 wherein the PEU is transferred from the conditional replication shuttle vector to the BBPAC through homologous recombination between the conditional replication shuttle vector and the BBPAC.

6. The method of claim 3 wherein the PEU is transferred from the conditional replication shuttle vector to the BBPAC by the addition of transposase to the host cell; wherein the PEU is positioned in between a pair of inverted transposon ends on the conditional replication shuttle vector; wherein the host cell, the BBPAC, or the conditional replication shuttle vector contain a nucleic acid encoding transposase; wherein the transcription of said nucleic acid is under the control of an inducible promoter; and wherein the addition of transposase to the host cell is achieved by adding an inducer of the inducible promoter to the host cell.

7. 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 and the PEU from the isolated cell of claim 4;

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

(c) culturing the eukaryotic cell; wherein when the PEU is operatively upstream of 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.

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

(a) preparing a cognate cDNA using the mRNA of claim 7 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.

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

(a) constructing a BBPAC with a vector containing the PEU and the trappable eukaryotic gene;

(b) transfecting the 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.

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

(a) preparing a cognate cDNA using the mRNA of claim 9 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.

11. A method of placing a eukaryotic promoter exon/intron unit (PEU) into a 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 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, and the first marker gene are linked together on the conditional replication shuttle vector such that when the PEU integrates into the BBPAC, the first marker gene does not remain linked to the integrated PEU; 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 placing the exon of the trappable eukaryotic gene operatively downstream of the PEU;

(b) growing the host cell under conditions in which the conditional replication shuttle vector can replicate, and a cell that contains the first and second marker genes is selected for; 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.

12. The method of claim 11 wherein the PEU does not contain an exon encoding a 3' polyadenylation sequence.

13. 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 obtained following step (d) of claim 11 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.

14. 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 13;

(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.

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

(a) preparing a cognate cDNA using the mRNA of claim 14 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.

16. A method of preparing a gene map for a BBPAC contig comprising:

(a) placing a eukaryotic promoter exon/intron unit (PEU) into the BBPACs that contain a trappable eukaryotic gene of a BBPAC contig by the method of claim 1; wherein the BBPAC contig contains BBPACs comprising overlapping trappable eukaryotic genes;

(b) isolating the BBPACs;

(c) transfecting the isolated BBPACs into eukaryotic cells;

(d) culturing the eukaryotic cells, 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;

(e) preparing cognate cDNAs using the mRNAs as a template; and

(f) assigning the physical location of each gene within the BBPAC by hybridization of the cognate cDNAs to the BBPACs of the BBPAC contig.

17. The method of claim 16 wherein the BBPAC is a BAC.

18. A method of constructing a cDNA library of cognate cDNAs to genomic DNA contained in a BBPAC genomic library comprising:

(a) sub-dividing a BBPAC genomic DNA library into individual BBPAC genomic sub-libraries;

(b) placing a eukaryotic promoter exon/intron unit (PEU) into the BBPACs that contain a trappable eukaryotic gene of a of a BBPAC sub-library by the method of claim 1; wherein the BBPACs of the BBPAC sub-library contain trappable eukaryotic genes;

(c) isolating the BBPACs;

(d) transfecting the isolated BBPACs into eukaryotic cells;

(e) culturing the eukaryotic cells, 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; and

(f) preparing a cDNA library of cognate cDNAs using the mRNAs as a template.

19. The method of claim 18 further comprising determining the nucleotide sequence of a cognate cDNA; wherein the nucleotide sequence of the trappable eukaryotic gene contained in the BBPAC is determined.

20. The method of claim 18 wherein the BBPAC genomic sub-library comprises 100-200 BBPACs.

21. The method of claim 18 wherein the genomic DNA library is a human genomic DNA library.

22. The method of claim 18 wherein the BBPAC genomic library is a BAC library.

Details for Patent 6,156,574

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

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