Homologous recombination in mammalian cells

• US 5,612,205 A
• Filed: 03/01/1993
• Issued: 03/18/1997
• Est. Priority Date: 08/29/1990
• Status: Expired due to Term

• Current Assignee: Pharming Intellectual Property B.V. (Pharming Group N.V.)
• Original Assignee: Genpharm International Inc (Bristol-Myers Squibb Company)
• Inventors: Kay, Robert M., Pieper, Frank, Berns, Anton, Strijker, Rein, Krimpenfort, Paul
• Primary Examiner(s): Ziska, Suzanne E.
• Application Number:
  US07/989,002
• Time in Patent Office: 1,478 Days
• Field of Search: 800/2, 435/172.3
• US Class Current: 435/463
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/206 of yeast origin, e.g. YAC, 2u
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/85 mammalian

Abstract

The invention relates to methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments and transgenic mammalian cells and transgenic non-human mammals produced by such methods.

Claims

1. A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragments homologously recombine in said cell to form a recombined DNA segment greater than 50 kb in length, said recombined DNA segment being integrated into the genome of said cell, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.

2. The method of claim 1, wherein said cell is a non-human zygote.

3. The method of claim 1, wherein said co-introducing step is performed by microinjection.

4. The method of claim 1, wherein said recombined DNA segment encodes a protein.

5. The method of claim 4, wherein said protein is a human immunoglobulin.

6. The method of claim 4, wherein said protein is human lactoferrin.

14. The method of claim 1, wherein the cell is murine.

15. The method of claim 14, wherein the cell is an embryonic stem cell.

7. A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragment homologously recombine in said cell to form a recombined DNA segment of greater than 50 kb integrated into the genome of said cell, and said DNA segment does not encode a positive selection marker which can be used to detect homologous recombination between said first and said second fragments, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.

8. A method for generating a DNA segment inside an embryonic cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragment homologously recombine in said embryonic cell to form a recombined DNA segment of greater than 50 kb, said recombined DNA segment being integrated into the genome of said embryonic cell, wherein said mammalian embryonic cell is a non-human zygote or a mouse ES cell.

9. The method of claim 8, wherein said embryonic cell is a non-human zygote.

10. A method for generating a DNA segment inside a cell comprising the step of introducing at least a first, a second, and a third DNA fragment, each fragment different from the others, into a recombination competent mammalian cell, said second fragment having two sequence portions, one of which has at least 80% sequence identity to a sequence portion of the first fragment, the other of which has at least 80% sequence identity to a portion of the third fragment, wherein said first, second and third DNA fragment homologously recombine in said cell to form a recombined DNA segment of greater than 50 kb, said recombined DNA segment being integrated into the genome of said cell, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.

11. The method of claim 10, wherein said cell is a non-human zygote.

12. The method of claim 10, wherein said introducing step is performed by microinjection.

13. The method of claim 10, wherein said recombined DNA segment encodes a protein.