METHODS AND MATERIALS FOR PRODUCING TRANSGENIC ANIMALS

• US 20100146655A1
• Filed: 07/16/2009
• Published: 06/10/2010
• Est. Priority Date: 07/16/2008
• Status: Active Grant

• Current Assignee: Recombinetics, Inc
• Original Assignee: Recombinetics, Inc
• Inventors: FAHRENKRUG, SCOTT C., CARLSON, DANIEL F., GEURTS, ARON M.
• Granted Patent: US 8,309,791 B2
• Time in Patent Office: Days
• US Class Current: 800/25
• CPC Class Codes: A01K 2217/058 due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
• CPC Class Codes: A01K 2217/203 Animal model comprising inducible/conditional expression system, e.g. hormones, tet
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/103 Ovine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/035 Animal model for multifactorial diseases
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61D 19/00 Instruments or methods for reproduction or fertilisation
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2015/8527 for producing animal models, e.g. for tests or diseases
• CPC Class Codes: C12N 2310/14 interfering N.A.
• 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 2800/90 Vectors containing a transposable element

Abstract

Transgenic artiodactyls are described as well as methods of making and using such artiodactyls.

Claims

1. A transgenic pig, the nucleated cells of which comprise a nucleic acid construct, said nucleic acid construct comprising a transcriptional unit comprising a regulatory region operably linked to a nucleic acid sequence encoding a small interfering RNA that reduces expression of porcine cystic fibrosis transmembrane conductance regulator (CFTR), wherein an inverted repeat of a transposon flanks each side of said transcriptional unit.

2. The transgenic pig of claim 1, wherein said regulatory region is a constitutive promoter.

3. The transgenic pig of claim 1, wherein said regulatory region is an inducible promoter.

4. The transgenic pig of claim 3, wherein said inducible promoter is regulated by tetracycline or doxycycline.

5. The transgenic pig of claim 1, wherein said transposon is selected from the group consisting of Sleeping Beauty, Tol2, PiggyBac, Frog Prince, Minos, and Hsmar1.

6. The transgenic pig of claim 1, said nucleic acid construct further comprising an insulator element flanking each side of said transcriptional unit.

7. The transgenic pig of claim 1, wherein said nucleic acid construct is CpG-methylated.

8. The transgenic pig of claim 1, wherein said small interfering RNA is a small hairpin RNA.

9. A method for making a transgenic pig, said method comprising:

a) introducing a transgenic pig cell into an enucleated pig oocyte to establish a combined cell, wherein said transgenic pig cell comprises a nucleic acid construct and a source of a transposase, said nucleic acid construct comprising a transcriptional unit, said transcriptional unit comprising a regulatory region operably linked to a nucleic acid sequence encoding a small interfering RNA that reduces expression of porcine cystic fibrosis transmembrane conductance regulator (CFTR), wherein an inverted repeat of a transposon flanks each side of said transcriptional unit;

b) producing a porcine embryo from said combined cell;

c) transferring said porcine embryo to a recipient female; and

d) allowing said porcine embryo to develop in the recipient female to produce said transgenic pig, the nucleated cells of which comprise said nucleic acid construct.

10. The method of claim 9, wherein said source of said transposase is said nucleic acid construct.

11. The method of claim 9, wherein said source of said transposase is a different nucleic acid construct.

12. The method of claim 9, wherein said transposon is selected from the group consisting of Sleeping Beauty, Tol2, PiggyBac, Frog Prince, Minos, and Hsmar1.

13. The method of claim 9, said nucleic acid construct further comprising an insulator element flanking each side of said transcriptional unit.

14. The method of claim 9, wherein said regulatory region is a constitutive promoter.

15. The method of claim 9, wherein said regulatory region is an inducible promoter.

16. The method of claim 15, wherein said inducible promoter is regulated by tetracycline or doxycycline.

17. The method of claim 9, wherein said small interfering RNA is a small hairpin RNA.

18. A method for making a transgenic artiodactyl, said method comprising:

a) injecting a nucleic acid construct and a source of a transposase into a pronuclear staged in vitro fertilized egg, said nucleic acid construct comprising a transcriptional unit, said transcriptional unit comprising a regulatory region operably linked to a target nucleic acid sequence, wherein an inverted repeat of a transposon flanks each side of said transcriptional unit;

b) transferring said injected fertilized egg to a recipient female; and

c) allowing said injected fertilized egg to develop in said recipient animal female to produce said transgenic artiodactyl.

19. The method of claim 18, wherein said source of said transposase is said nucleic acid construct.

20. The method of claim 18, wherein said source of said transposase is a different nucleic acid construct.

21. The method of claim 18, wherein said transposon is selected from the group consisting of Sleeping Beauty, Tol2, PiggyBac, Frog Prince, Minos, and Hsmar1.

22. The method of claim 18, said nucleic acid construct further comprising an insulator element flanking each side of said transcriptional unit.

23. The method of claim 18, wherein said regulatory region is a constitutive promoter.

24. The method of claim 18, wherein said regulatory region is a tissue-specific promoter.

25. The method of claim 24, wherein said tissue-specific promoter preferentially expresses in heart tissue or θ

-cells.

26. The method of claim 18, wherein said regulatory region is inducible.

27. The method of claim 26, wherein said inducible regulatory region is regulated by tetracycline or doxycycline.

28. The method of claim 18, wherein said target nucleic acid sequence encodes a polypeptide.

29. The method of claim 28, wherein said polypeptide is a recombinase.

30. The method of claim 28, wherein said polypeptide is a fluorescent polypeptide.

31. The method of claim 18, wherein said target nucleic acid sequence encodes a small interfering RNA.

32. The method of claim 31, wherein said small interfering RNA is a small hairpin RNA.

33. The method of claim 32, wherein said small hairpin RNA reduces expression of cystic fibrosis transmembrane conductance regulator (CFTR).

34. The method of claim 26, wherein said artiodactyl is a pig and said target nucleic sequence encodes a small hairpin RNA that reduces expression of porcine CFTR.

35. A method for making a transgenic artiodactyl, said method comprising:

a) injecting at least two nucleic acid constructs and a source of a transposase into a pronuclear staged in vitro fertilized egg, wherein each said nucleic acid construct comprises a transcriptional unit and an inverted repeat of a transposon flanking each side of said transcriptional unit, wherein said transcriptional unit of one of said nucleic acid construct comprises a regulatory region operably linked to a target nucleic acid sequence, and wherein said transcriptional unit of one of said nucleic acid constructs comprises a regulatory region operably linked to a nucleic acid sequence encoding a selectable marker;

b) transferring said injected fertilized egg to a recipient female; and

c) allowing said injected fertilized egg to develop in said recipient female to produce said transgenic artiodactyl.

36. The method of claim 35, wherein said nucleic acid construct encoding said selectable marker is provided in excess of said nucleic acid construct comprising said target nucleic acid sequence.

37. The method of claim 35 further comprising assaying the artiodactyl for presence of the marker and sexually breeding the artiodactyl to produce offspring that expresses one of the nucleic acid constructs and does not have the expressible selectable marker.

38. A transgenic artiodactyl, the nucleated cells of which comprise a nucleic acid construct, said nucleic acid construct comprising a transcriptional unit comprising a regulatory region operably linked to a nucleic acid sequence encoding a small interfering RNA that reduces expression of a target gene.

39. The transgenic artiodactyl of claim 38, wherein said small interfering RNA is placed with a transposon selected from the group consisting of Sleeping Beauty, Tol2, PiggyBac, Frog Prince, Minos, and Hsmar1.

40. The transgenic pig of claim 38, wherein said small interfering RNA is a small hairpin RNA and the target gene is cystic fibrosis transmembrane conductance regulator (CFTR).

41. A nucleic acid construct comprising a hypermethylated transposon.

42. The construct of claim 41 comprising a plasmid that comprises the hypermethylated transposon.