Transgenesis with humanized immunoglobulin loci

• US 20050153392A1
• Filed: 08/10/2004
• Published: 07/14/2005
• Est. Priority Date: 08/11/2003
• Status: Abandoned Application

• Current Assignee: Therapeutic Human Polyclonals, Inc., (Roche Holding AG)
• Original Assignee: Therapeutic Human Polyclonals, Inc., (Roche Holding AG)
• Inventors: Buelow, Roland, van Schooten, Wim
• Application Number:
  US10/916,082
• Publication Number:
  US 20050153392A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/107 Rabbit
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/204 of bacterial origin, e.g. BAC
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

The invention concerns recombinase-mediated transfer of human or humanized immunoglobulin loci into the genome of non-human transgenic animals. In particular, the invention relates to improved methods to integrate human and/or humanized immunoglobulin loci into the genome of non-human animals using transgenic constructs which contain immunoglobulin loci comprising human gene sequences and a site recognized by a site specific recombinase which can be used to catalyze the insertion of the transgene into the animal'"'"'s genome.

Claims

1. An isolated nucleic acid molecule comprising one or more human or humanized immunoglobulin loci and at least one recombination site.

2. The isolated nucleic acid molecule of claim 1, wherein said recombination site is the substrate for a recombinase selected from the group consisting of Cre-recombinase, Cre-like recombinase, FLP recombinase, and R recombinase.

3. The isolated nucleic acid molecule of claim 1, wherein said recombination site is the substrate for a transposase or a retrotransposase.

4. The isolated nucleic acid molecule of claim 2, which is circular.

5. The isolated nucleic acid molecule of claim 3, which is linear or circular.

6. A method for the site specific integration of a transgene comprising one or more human or humanized immunoglobulin loci in the genome of a eukaryotic cell, said method comprising:

introducing (i) a targeting construct comprising one or more human or humanized immunoglobulin loci and a first recombination site, and (ii) a site specific recombinase into a eukaryotic cell, wherein the genome of said cell comprises a second recombination site, maintaining the eukaryotic cell under conditions that allow recombination between the first and second recombination sites, wherein the recombination is mediated by the site specific recombinase and the result of the recombination is site specific integration of one or more human or humanized immunoglobulin loci in the genome of the eukaryotic cell.

7. The method of claim 6 wherein the recombinase is other than a transposase or a retrotransposase.

8. The method of claim 7, wherein said targeting construct is circular.

9. The method of claim 6, wherein the recombinase is a transposase or a retrotransposase.

10. The method of claim 9, wherein the targeting construct is linear or circular.

11. The method of claim 7, wherein the site-specific recombinase is a recombinase expressed by a phage.

12. The method of claim 11, wherein the phage is selected from the group consisting of (C31 integrase, TP901-1 and R4.

13. The method of claim 6, wherein the site-specific recombinase is a recombinase selected from the group consisting of Cre-recombinase, Cre-like recombinase, FLP recombinase, and R recombinase.

14. The method of claim 6, wherein the first and second recombination sites share at least 90% sequence identity.

15. The method of claim 6, wherein the first and second recombination sites share less than 90% sequence identity.

16. The method of claim 6, wherein the recombinase facilitates recombination between a bacterial genomic recombination site (attB) and a phage recombination site (attP).

17. The method of claim 16, wherein the first recombination site comprises an attB site, and the second recombination site comprises a pseudo-attP site.

18. The method of claim 16, wherein the first recombination site comprises an pseudo-attB site, and the second recombination site comprises an attP site.

19. The method of claim 17 or claim 18, wherein the recombinase is encoded by Φ

C31 or phage R4 or TP901-1.

20. The method of claim 16, wherein the recombinase-facilitated recombination results in a site that is no longer a substrate for the recombinase.

21. The method of claim 16, wherein said recombinase is introduced into the cell as a polypeptide.

22. The method of claim 16, wherein said recombinase is introduced into the cell as a messenger RNA molecule encoding a recombinase polypeptide.

23. The method of claim 16, wherein said recombinase is introduced into the cells as an expression cassette encoding the recombinase polypeptide.

24. The method of claim 16, wherein said recombinase in introduced into the cell before introduction of said construct comprising one or more human or humanized immunogloblin loci.

25. The method of claim 16, wherein said recombinase in introduced into the cell concurrently with introduction of said construct comprising one or more human or humanized immunogloblin loci.

26. The method of claim 16, wherein said recombinase in introduced into the cell after introduction of said construct comprising one or more human or humanized immunogloblin loci.

27. A eukaryotic cell comprising one or more human or humanized immunoglobulin loci whose integration into the cellular genome was mediated by a recombinase.

28. A transgenic animal comprising at least one cell according to claim 27.