Modulation of CRE recombinase in the in vivo cloning of DNA

• US 5,434,066 A
• Filed: 03/15/1994
• Issued: 07/18/1995
• Est. Priority Date: 01/24/1992
• Status: Expired due to Fees

• Current Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Hartley, James L., Bebee, Robert L.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/214,023
• Time in Patent Office: 490 Days
• Field of Search: 435/172.3, 435/252.3, 435/252.33, 435/320.1
• US Class Current: 435/475
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Methods and recombinant vectors suitable for accomplishing the in vivo alteration of a nucleic acid molecule are disclosed. The invention in particular discloses the use of recombinases such as Cre to accomplish in vivo recombination.

Claims

1. A method for accomplishing in vivo site-specific recombination between Lox sites of a target molecule in a host cell which comprises the steps:

(A) providing to said host cell a first vector, said vector containing (i) a Cre recombinase gene and (ii) a replicon, wherein said Cre recombinase gone is capable of being expressed in said host cell and said replicon is sufficient to permit repliCatiOn of said first vector;

(B) providing tO said host cell a second vector, said second vector containing (i) said target molecule and lox sites, (ii) a replicon, and (iii) a determinant that has the capacity tO inhibit the expression of siad Cre recombinase oene of said first vector;

wherein said inhibition is irreversible and;

(C) culturing said host cell under conditions sufficient to permit the expression of said Cre recombinase gone, to thereby mediate the in vivo site-specific recombination of the lox sites of said second vector, said site-specific recombination causing the excision of said target molecule from said second vector;

wherein said second vector inhibits expression of said Cre recombinase, and, thereby results in the modulation of the expression of said Cre recombinase gene.

2. The method of claim 1, wherein said determinant of said second vector inhibits the expression of said Cre recombinase gens on the first vector by expressing a gens present on said second vector.

3. The method of claim 1, wherein said determinant of said second vector comprises an incompatibility determinant sufficient to inhibit the replication of said first vector and cause the loss of said first vector from the said host cell.

4. The method of claim 3, wherein said replicon of said first vector is a P1 replicon that lacks at least one naturally present incA regulatory element, and wherein said second vector contains an incA regulatory element, such that the presence of said second vector inhibits the replication of said first vector.

5. A host cell containing a first and second vector, wherein said first vector contains (i) a Cre recombinase gene and (ii) a replicon wherein said Cre recombinase gens is capable of being expressed in said host cell and said replicon is sufficient to permit replication of said first vector;

and wherein said second vector contains (i) a target moleCUle containing lox sites recognized by said Cre recombinase gene, (ii) a replicon, and (iii) a determinant that has the capacity to inhibit the expression of said Cre recombinase gene of said first vector wherein said determinant of said second vector comprises an incompatibility determinant sufficient to inhibit the replication of said first vector.

6. The host cell of claim 5, wherein said replicon of said first vector is a P1 replicon, and wherein said incompatibility determinant comprises an incA regulatory element.

7. The host cell of claim 6, wherein said P1 replicon of said first vector lacks at least one naturally present incA regulatory element.