Conditionally amplifiable BAC vector

• US 5,874,259 A
• Filed: 11/21/1997
• Issued: 02/23/1999
• Est. Priority Date: 11/21/1997
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Szybalski, Waclaw
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Yucel, Irem
• Application Number:
  US08/975,763
• Time in Patent Office: 459 Days
• Field of Search: 435/320.1, 435/91.1, 435/252.33
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

A system for obtaining large amounts of a genomic DNA fragment from a bacterial artificial chromosome includes a vector that has a site into which the genomic fragment can be cloned and, flanking the site are excision mediating sites. The vector also includes, between the excision mediating sites and near one of the sites, a controllable origin of replication.

Claims

1. A bacterial artificial chromosome comprising

a site into which an DNA fragment can be cloned;

a pair of inducible excision-mediating sites flanking the site into which the DNA fragment can be cloned, the excision-mediating sites being provided in parallel orientation relative to one another and defining an excisable fragment that comprises the site into which the DNA fragment can be cloned; and

an inducible origin of replication on the excisable fragment.

2. A bacterial artificial chromosome as claimed in claim 1 wherein the pair of excision-mediating sites are FRT sites.

3. A bacterial artificial chromosome as claimed in claim 1 wherein the pair of excision-mediating sites comprise a sequence as shown in SEQ ID NO:

3.

4. A bacterial artificial chromosome as claimed in claim 1 wherein the inducible origin of replication is oriV.

5. A cell comprising in its interior a bacterial artificial chromosome that comprises a site into which an DNA fragment can be cloned, a pair of inducible excision-mediating sites flanking the site into which the DNA fragment can be cloned, the excision-mediating sites being provided in parallel orientation relative to one another and defining an excisable fragment that comprises the site into which the DNA fragment can be cloned, and an inducible origin of replication on the excisable fragment;

a gene encoding an excision-mediating protein that induces the excisable fragment to excise from the bacterial artificial chromosome and circularize to form an excised plasmid; and

a gene encoding an amplification-mediating protein that induces the excised plasmid to replicate.

6. A cell as claimed in claim 5 wherein the gene encoding the excision-mediating protein is a FLP gene.

7. A cell as claimed in claim 5 wherein the gene encoding the amplification-mediating protein is selected from a group consisting of a trfA gene and a mutant thereof that encodes an amplication-mediating protein.

8. A method for amplifying in a cell an insert in a bacterial artificial chromosome, the insert being provided on an excisable fragment between a pair of inducible excision-mediating sites, the excisable fragment further comprising an inducible origin of replication, the method comprising the steps of:

providing in the host cell an excision-mediating protein that induces the excisable fragment to excise from the bacterial artificial chromosome and to circularize to form an excised plasmid; and

providing in the host cell an amplification-mediating protein that induces the excised plasmid that comprises the insert to replicate.

9. A bacterial artificial chromosome comprising an inducible origin of replication.