Methods for producing genetically modified animals using hypermethylated transposons

• US 8,785,718 B2
• Filed: 10/05/2012
• Issued: 07/22/2014
• 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.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/645,788
• Publication Number:
  US 20130090522A1
• Time in Patent Office: 655 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

Methods of using hypermethylated transposons to create genetically modified animals that express interfering RNAs are described.

Claims

1. A method of making a nonhuman transgenic animal whose genome comprises a hypermethylated transposon construct comprising

hypermethylating a transposon, introducing the hypermethylated transposon comprising a transcriptional unit comprising a regulatory region operably linked to an interfering RNA (RNAi) encoding nucleic acid into a nonhuman animal egg, said transposon being inserted into a genome of the egg, and

allowing the egg to develop in a recipient female to produce the nonhuman transgenic animal, wherein the nonhuman animal expresses the RNAi sequence.

2. The method of claim 1 wherein the animal is an artiodactyl.

3. The method of claim 1 comprising preparing the egg with the in vitro hypermethylated transposon and transferring said egg to the recipient female.

4. The method of claim 1 wherein introducing the hypermethylated transposon into the nonhuman animal egg comprises introduction of the transposon and a transposase into a single-cell fertilized egg, with the transposase inserting the transposon into the genome.

5. The method of claim 1 wherein introducing the hypermethylated transposon into the nonhuman animal egg comprises enucleation of the egg followed by transfer of a nucleus of a somatic cell into the egg, with the transferred nucleus comprising the gene and the transposon inserted into the genome.

6. The method of claim 1 wherein introducing the hypermethylated transposon into the nonhuman animal egg comprises transfer of chromatin of a somatic pig cell into the egg, with the chromatin of the somatic cell comprising the transposon.

7. The method of claim 1 wherein all cytosine-phosphodiester-guanine sites in the transposon are methylated.

8. The method of claim 1 wherein at least 90% of cytosine-phosphodiester-guanine sites in the transposon are methylated.

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

10. The method of claim 1 comprising a plasmid that comprises the hypermethylated transposon.

11. The method of claim 1, wherein said RNAi is a small hairpin RNA.

12. The method of claim 1 wherein the target gene is cystic fibrosis transmembrane conductance regulator (CFTR).