Animal models of atherosclerosis
The present invention provides transgenic, large non-human animal models of atherosclerosis and hypercholesterolemia, as well as methods of using such animal models in the identification and characterization of therapies for atherosclerosis and hypercholesterolemia.
1. A transgenic Yucatan miniature pig whose genome comprises a homozygous inactivation of exon 4 in its endogenous low-density lipoprotein receptor (LDLR) gene, wherein said exon 4 comprises insertion of a gene targeting vector comprising the nucleotide sequence as set forth in SEQ ID NO: 3, wherein said pig produces no functional LDLR protein, and wherein said pig exhibits an increased level of triglycerides, LDL and VLDL cholesterol as compared to a wild type Yucatan miniature pig.
2. A transgenic Yucatan miniature pig whose genome comprises a heterozygous inactivation of exon 4 in its endogenous LDLR gene, wherein said exon 4 comprises insertion of a gene targeting vector comprising the nucleotide sequence as set forth in SEQ ID NO: 3, wherein said pig shows reduced expression of LDLR gene and an increased level of and VLDL cholesterol as compared to a wild type Yucatan miniature pig.
3. A method of making the Yucatan miniature pig of claim 2 , the method comprising:
(a) inactivation by inserting a heterologous polynucleotide comprising the nucleic acid sequence of SEQ ID NO:3 into exon 4 of the Yucatan miniature pig LDLR gene via homologous recombination in a Yucatan miniature pig fetal fibroblast;
(b) culturing the fetal fibroblast in a medium that selects for the neomycin-resistance gene for less than 20 days;
(c) introducing the nucleus of the fetal fibroblast into an enucleated Yucatan miniature pig oocyte to form a Yucatan miniature pig embryo; and
(d) transferring the Yucatan miniature pig embryo into a recipient female Yucatan miniature pig and allowing the embryo to develop such that a heterozygous transgenic Yucatan miniature pig of claim 2 whose genome comprises a heterozygous disruption of the LDLR gene is obtained.
4. The method of claim 3 , wherein the fetal fibroblast is from a male pig.
5. The method of claim 3 , wherein the fetal fibroblast is from a female pig.
6. The method of claim 3 further comprising the step of:
(e) breeding the heterozygous transgenic swine obtained in (d) such that a homozygous Yucatan miniature pig of claim 1 is obtained.
7. The method of claim 6 , wherein the fetal fibroblast is from a male pig.
8. The method of claim 6 , wherein the fetal fibroblast is from a female pig.