Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same

• US 5,631,153 A
• Filed: 06/05/1995
• Issued: 05/20/1997
• Est. Priority Date: 08/22/1989
• Status: Expired due to Term

• Current Assignee: University of Utah (State of Utah)
• Original Assignee: University of Utah (State of Utah)
• Inventors: Capecchi, Mario R., Thomas, Kirk R.
• Primary Examiner(s): Campell, Bruce R.
• Application Number:
  US08/463,813
• Time in Patent Office: 715 Days
• Field of Search: 435/172.3, 435/320.1, 435/240.1, 435/240.2, 435/240.4, 935/22, 935/56
• US Class Current: 435/6.14
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/1077 Pentosyltransferases (2.4.2)

Abstract

Positive-negative selector (PNS) vectors are provided for modifying a target DNA sequence contained in the genome of a target cell capable of homologous recombination. The vector comprises a first DNA sequence which contains at least one sequence portion which is substantially homologous to a portion of a first region of a target DNA sequence. The vector also includes a second DNA sequence containing at least one sequence portion which is substantially homologous to another portion of a second region of a target DNA sequence. A third DNA sequence is positioned between the first and second DNA sequences and encodes a positive selection marker which when expressed is functional in the target cell in which the vector is used. A fourth DNA sequence encoding a negative selection marker, also functional in the target cell, is positioned 5'"'"' to the first or 3'"'"' to the second DNA sequence and is substantially incapable of homologous recombination with the target DNA sequence. The invention also includes transformed cells containing at least one predetermined modification of a target DNA sequence contained in the genome of the cell. In addition, the invention includes organisms such as non-human transgenic animals and plants which contain cells having predetermined modifications of a target DNA sequence in the genome of the organism.

Claims

1. A positive-negative selection (PNS) vector comprising:

a first homologous vector DNA sequence capable of homologous recombination with a first region of a target DNA sequence native to the genome of target cells,

a positive selection marker DNA sequence capable of conferring a positive selection characteristic in said cells,

a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence, and

a negative selection marker DNA sequence, capable of conferring a negative selection characteristic in said cells, thereby allowing killing of said cells, but substantially incapable of homologous recombination with said target DNA sequence,

wherein the spatial order of said sequences in said PNS vector is;

said first homologous vector DNA sequence, said positive selection marker DNA sequence, said second homologous vector DNA sequence and said negative selection marker DNA sequence as shown in FIG. 1,

wherein the 5'"'"'-3'"'"' orientation of said first homologous vector sequence relative to said second homologous vector sequence is the same as the 5'"'"'-3'"'"' orientation of said first region relative to said second region of said target sequence;

wherein the vector is capable of modifying said target DNA sequence by homologous recombination of said first homologous vector DNA sequence with said first region of said target sequence and of said second homologous vector DNA sequence with said second region of said target sequence.

2. The PNS vector of claim 1 wherein said target DNA contains exons and introns and said positive selections marker DNA sequence further contains the exon-intron and intron-exon splicing sequences for an intron in said target DNA.

3. The PNS vector of claim 2 wherein said first or said second homologous vector DNA sequence contains at least a portion of said exon in said target DNA sequence wherein one or more nucleotides have been substituted, deleted or inserted.

4. The PNS vector of claim 1 wherein said target DNA sequence contains exons and introns and said first and homologous vector DNA sequences contain different portions of the same exon in said target DNA sequence.

5. The PNS vector of claim 1 wherein said positive selection marker DNA sequence further comprises a second negative selection marker DNA sequence different from the negative selection marker DNA sequence downstream from said second homologous vector DNA sequence.

6. The PNS vector of claim 1 wherein said PNS vector has a length between 20 kb and 50 kb.

7. The PNS vector of claim 1 wherein said homologous portions in said first and said second homologous vector DNA sequences have a length between 1,000 base pairs and 15,000 base pairs each.

8. The PNS vector of claim 1 where said negative selection marker DNA sequence is selected from group consisting of DNA sequences encoding Hprt, gpt, HSV-tk, diphtheria toxin, ricin toxin and cytosine deaminase.

9. The PNS vector of claim 1 wherein said first or said second homologous vector DNA sequence further comprises a modifying DNA sequence encoding a modification of said target DNA sequence.

10. The PNS vector of claim 1 wherein said first or said second homologous vector DNA sequence encodes the correction of a genetic defect in said target DNA.

11. The PNS vector of claim 1 wherein said target cells are derived from a multicellular organism and are capable of homologous recombination.

12. The PNS vector of claim 1 wherein said target cells are plant cells capable of homologous recombination.

13. The PNS vector of claim 1 wherein said target cells are animal cells capable of homologous recombination.

14. The PNS vector of claim 13 wherein said target cells are selected from the group consisting of hematopoietic, epithelial, liver, lung, bone, endothelial, mesenchymal, neural and muscle stem cells.

15. The PNS vector of claim 1 further comprising an additional negative selection marker DNA sequence upstream from said first homologous vector DNA sequence, wherein said additional negative selection marker DNA sequence is capable of conferring a negative selection characteristic in said cells, thereby allowing killing of said cells, but is substantially incapable of homologous recombination with said target DNA sequence.

16. A method for selecting a transformed cell containing a modification in a target DNA sequence in the genome of said cell comprising:

(a) transfecting cells capable of mediating homologous recombination with a positive-negative selection vector comprising;

a first homologous vector DNA sequence capable of homologous recombination with a first region of said target DNA sequence,

a positive selection marker DNA sequence capable of conferring a positive selection characteristic in said cells,

a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence, and

a negative selection marker DNA sequence, capable of conferring a negative selection characteristic in said cells, but substantially incapable of homologous recombination with said target DNA sequence,

wherein the spatial order of said sequences in said PNS vector is;

said first homologous vector DNA sequence, said positive selection marker DNA sequence, said second homologous vector DNA sequence and said negative selection marker DNA sequence as shown in FIG. 1,

wherein the 5'"'"'-3'"'"' orientation of said first homologous vector sequence relative to said second homologous vector sequence is the same as the 5'"'"'-3'"'"' orientation of said first region relative to said second region of said target sequence;

wherein the vector is capable of modifying the target DNA sequence by homologous recombination of said first and second homologous vector sequences with the first and second regions of said target sequence;

(b) selecting for transformed cells in which said positive-negative selection vector has integrated into said target DNA sequence by homologous recombination by sequentially or simultaneously selecting against transformed cells containing said negative selection marker and selecting for cells containing said positive selection marker, and

(c) analyzing the DNA of transformed cells surviving the selection step to identify a cell containing the modification.

17. The method of claim 16, further comprising introducing a second modification into the genome of said cell or a cell propagated therefrom.

18. The method of claim 16 wherein said cells are derived from a multicellular organism and are capable of homologous recombination.

19. The method of claim 16 wherein said cells are plant cells capable of homologous recombination.

20. The method of claim 16 wherein said cells are animal cells capable of homologous recombination.

21. The method of claim 20 wherein said cells are selected from the group consisting of hematopoietic, epithelial, liver, lung, bone marrow, endothelial, mesenchymal, neural and muscle stem cells.

22. The method of claim 21, wherein said cells are human cells.

23. The method of claim 21, wherein said cells are bone marrow stem cells.

24. The method of claim 16, wherein said cells are simian cells.

25. The method of claim 16, wherein said cells are human cells.