Title:
MITOCHONDRIAL-NUCLEAR EXCHANGED CELLS, TISSUES, ORGANS AND ANIMALS
Kind Code:
A1


Abstract:
Provided herein are mitochondrial-nuclear exchanged cells and animals comprising mitochondrial DNA (mtDNA) from one subject and nuclear DNA (nDNA) from a different subject. Methods for producing a mitochondrial-nuclear exchanged animal and animals made by the methods are provided. Also provided are methods of screening for agents useful for treating a disease or disorder using mitochondrial-nuclear exchanged animals or cells, tissues or organs thereof.



Inventors:
Ballinger, Scott Webster (Odenville, AL, US)
Welch, Danny R. (Vestavia Hills, AL, US)
Kesterson, Robert Allen (Birmingham, AL, US)
Johnson, Larry W. (Jasper, AL, US)
Application Number:
12/486375
Publication Date:
12/31/2009
Filing Date:
06/17/2009
Assignee:
The UAB Research Foundation (Birmingham, AL, US)
Primary Class:
Other Classes:
435/325, 800/13, 800/18, 800/24, 435/29
International Classes:
G01N33/00; A01K67/00; C12N5/00; C12N15/00; C12Q1/02
View Patent Images:



Primary Examiner:
CROUCH, DEBORAH
Attorney, Agent or Firm:
Meunier Carlin & Curfman LLC (999 Peachtree Street NE Suite 1300, Atlanta, GA, 30309, US)
Claims:
What is claimed is:

1. A cell comprising mitochondrial DNA (mtDNA) from a subject susceptible to a disease or disorder and nuclear DNA (NDNA) from a subject that is not susceptible to the disease or disorder.

2. The cell of claim 1, wherein the nDNA is from a wild-type subject.

3. The cell of claim 1, wherein the nDNA is from a subject resistant to the disease or disorder.

4. The cell of claim 1, wherein the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder.

5. The cell of claim 1, wherein the cell is an oocyte.

6. The cell of claim 1, wherein the cell is an embryonic cell.

7. A zygote or an embryo comprising the cell of claim 1.

8. The embryo of claim 7, wherein the embryo is a pro-nuclear embryo.

9. A chimeric animal comprising a plurality of cells of claim 1.

10. The chimeric animal of claim 9, wherein the animal further comprises a mutation in a gene associated with the disease or disorder.

11. The chimeric animal of claim 10, wherein the gene is not expressed or the protein expressed by the gene is non-functional.

12. The chimeric animal of claim 9, wherein the animal is a mouse comprising mtDNA from C57BL/6J mice and nDNA from C3H/HeN mice.

13. The chimeric animal of claim 9, wherein the animal is a female.

14. A progeny animal of the animal of claim 9.

15. A progeny animal resulting from a cross between the chimeric animal of claim 13 and a knockout mouse, wherein the knockout mouse comprises a mutation in at least one gene associated with the disease or disorder such that the gene is not expressed or the protein expressed by the gene is not functional.

16. The progeny animal of claim 15, wherein the knockout mouse is susceptible to the disease or disorder.

17. A method of screening for agents useful for treating a disease or disorder comprising: (a) administering to the animal of claim 9 an agent to be tested; and (b) determining whether the agent prevents or reduces one or more symptoms of the disease or disorder.

18. A cell comprising mitochondrial DNA (mtDNA) from a subject resistant to a disease or disorder and nuclear DNA (nDNA) from a wild-type subject or a subject that is susceptible to the disease or disorder.

19. The cell of claim 18, wherein the NDNA is from a wild-type subject.

20. The cell of claim 18, wherein the NDNA is from a subject susceptible to the disease or disorder.

21. The cell of claim 18, wherein the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder.

22. The cell of claim 18, wherein the cell is an oocyte.

23. The cell of claim 18, wherein the cell is an embryonic cell.

24. A zygote or an embryo comprising the cell of claim 18.

25. The embryo of claim 24, wherein the embryo is a pro-nuclear embryo.

26. A chimeric animal comprising a plurality of cells of claim 18.

27. The chimeric animal of claim 26, wherein the animal is a mouse comprising mtDNA from NZB/B1NJ mice and nDNA from FVB/N-TgN(MMTVPyMT) mice.

28. A method for producing a mitochondrial-nuclear exchanged animal comprising: (a) selecting an animal susceptible to a disease or disorder; (b) selecting an animal that is not susceptible to the disease or disorder; (c) harvesting a pro-nuclear embryo from each of the animals of steps (a) and (b); (d) enucleating the embryos of step (c); (e) transferring the nucleus from the embryo of the animal that is not susceptible to the disease or disorder to the enucleated embryo of the animal susceptible to the disease or disorder to make a resulting embryo, wherein the resulting embryo has mtDNA from the animal susceptible to the disease or disorder and the nDNA from the animal that is not susceptible to the disease or disorder; and (f) transferring the resulting embryo into an appropriate host and allowing the transferred embryo to develop into a progeny animal, wherein the progeny animal is a mitochondrial-nuclear exchanged animal.

29. The method of claim 28, wherein the animal that is not susceptible to the disease or disorder is from a wild-type animal or an animal resistant to the disease or disorder.

30. The method of claim 28, further comprising selecting a female mitochondrial-nuclear exchanged animal.

31. A method of generating progeny of the female mitochondrial-nuclear exchanged animal of claim 30 by crossing the female mitochondrial exchanged animal with a knockout animal, wherein the knockout animal comprises a mutation in at least one gene associated with the disease or disorder such that the gene is not expressed or the protein expressed by the gene is not functional.

32. The method of claim 31, further comprising selecting progeny animals of the cross that comprise mtDNA from the mitochondrial-nuclear exchanged animals and nDNA from the knockout mouse.

33. The method of claim 28, wherein the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder.

34. A mitochondrial-nuclear exchanged animal made by the method of claim 28.

35. A method of screening for agents useful for treating a disease or disorder comprising the steps of: (a) providing a mitochondrial-nuclear exchanged animal comprising mtDNA from an animal susceptible to the disease or disorder and nDNA from an animal not susceptible to the disease or disorder; (b) administering to the animal an agent to be tested; and (c) determining whether the agent prevents or reduces one or more symptoms of the disease or disorder.

36. The method of claim 35, wherein the animal not susceptible to the disease or disorder is a wild-type animal or an animal resistant to the disease or disorder.

37. The method of claim 35, wherein the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder.

38. The method of claim 37, wherein the disease is cancer and wherein the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from a mouse selected from the group consisting of FVB/N-TgN(MMTVPyMT), AKR/J, and A/J mice.

39. The method of claim 37, wherein the disease is cardiovascular disease and wherein the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from C57BL/6J or DBA/2J mice.

40. The method of claim 35, wherein the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from C57BL/6J mice and NDNA from C3H/HeN mice.

41. The method of claim 35, wherein the mitochondrial-nuclear exchanged animal is a mouse comprising NDNA from a mouse selected from the group consisting of C57BL/6J, 129, A/J, BALB/c or C3H/HeN mice.

42. A method of screening for agents useful for treating a disease or disorder comprising the steps of: (a) providing a mitochondrial-nuclear exchanged cell, tissue or organ comprising mtDNA from an animal susceptible to the disease or disorder and nDNA from an animal not susceptible to the disease or disorder; (b) contacting the cell, tissue or organ with an agent to be tested; and (c) determining whether the agent prevents or reduces one or more symptoms of the disease or disorder.

43. The method of claim 42, wherein the cells, tissues or organs are obtained from a mitochondrial-nuclear exchanged animal or progeny thereof.

44. The method of claim 42, wherein the animal not susceptible to the disease or disorder is a wild-type animal or an animal resistant to the disease or disorder.

45. The method of claim 42, wherein the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder.

46. The method of claim 43, wherein the disease is cancer and wherein the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from a mouse selected from the group consisting of FVB/N-TgN(MMTVPyMT), AKR/J, and A/J mice.

47. The method of claim 43, wherein the disease is cardiovascular disease and wherein the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from C57BL/6J or DBA/2J mice.

48. The method of claim 43, wherein the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from C57BL/6J mice and nDNA from C3H/HeN mice.

49. The method of claim 43, wherein the mitochondrial-nuclear exchanged animal is a mouse comprising nDNA from a mouse selected from the group consisting of C57BL/6J, 129, A/J, BALB/c or C3H/HeN mice.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional Application No. 61/076,260, filed Jun. 27, 2008, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No. W91XWH-07-1-0540 awarded by the Department of Defense. The government has certain rights in the invention.

BACKGROUND

Each cell contains hundreds of mitochondria and thousands of mitochondrial DNA (mtDNA) copies, which are maternally inherited. The mammalian mtDNA encodes 13 polypeptides that are essential for oxidative phosphorylation (OXPHOS) plus two rRNAs (12S and 16S) and 22 tRNAs that are required for mitochondrial protein synthesis. The mtDNA encoded polypeptide genes are structural subunits for four of the five OXPHOS enzyme complexes (I, III, IV and V). The nuclear DNA (nDNA) codes for all other mitochondrial proteins including all four subunits of complex II (succinate dehydrogenase), the mitochondrial DNA polymerase y subunits, the mitochondrial RNA polymerase components, the mitochondrial transcription factor (mtTFA), the mitochondrial ribosomal proteins and elongation factors, and the mitochondrial metabolic enzymes. Mitochondria generate energy via OXPHOS which “couples” electron transport with proton translocation for the production of ATP. Mitochondria are also the primary source of endogenous cellular ROS. The efficacy of mitochondrial energy and oxidant production is dependent upon a number of factors including local concentrations of both reactive nitrogen and oxygen species, mitochondrial antioxidants, cytokines, electron transport efficiency, metabolic reducing equivalent availability (NADH and FADH2), uncoupling protein (UCP) activities, and overall organelle integrity (damage to membranes, DNA, and proteins).

SUMMARY

Provided herein are cells and animals comprising mitochondrial DNA (mtDNA) from a subject susceptible to a disease or disorder and nuclear DNA (nDNA) from a subject that is not susceptible to the disease or disorder. Also provided are cells and animals comprising mitochondrial DNA (mtDNA) from a subject resistant to a disease or disorder and nuclear DNA (nDNA) from a wild-type subject or a subject that is susceptible to the disease or disorder. Such animals are referred to herein as mitochondrial-nuclear exchanged animals. Also provided are progeny of mitochondrial-nuclear exchanged animals and progeny animals resulting from a cross between the mitochondrial-nuclear exchanged animals and a knockout mouse, wherein the knockout mouse comprises a mutation in at least one gene associated with the disease or disorder such that the gene is not expressed or the protein expressed by the gene is not functional.

Methods for producing a mitochondrial-nuclear exchanged animal and animals made by the methods are provided. The method comprises selecting an animal susceptible to a disease or disorder, selecting an animal that is not susceptible to the disease or disorder, harvesting pro-nuclear embryos from the animals, enucleating the embryos, transferring the nucleus from the animal that is not susceptible to the disease or disorder to the enucleated embryo of the animal susceptible to the disease or disorder, wherein the embryo has mtDNA from the animal susceptible to the disease or disorder and the NDNA from the animal that is not susceptible to the disease or disorder, and transferring the embryo into an appropriate host and allowing the transferred embryo to develop into a progeny animal, wherein the progeny animal is a mitochondrial-nuclear exchanged animal.

Also provided is a method of screening for agents useful for treating a disease or disorder comprising the steps of providing a mitochondrial-nuclear exchanged animal comprising mtDNA from an animal susceptible to the disease or disorder and nDNA from an animal not susceptible to the disease or disorder, administering to the animal an agent to be tested, and determining whether the agent prevents or reduces one or more symptoms of the disease or disorder.

The details of one or more aspects are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the number of mitochondrial-nuclear exchanges performed resulting in term births, and surviving animals (6 weeks of age). Labels on the X-axis indicate mtDNA haplotype/nuclear genotype. PyMT refers to the FVB/N-TgN(MMTVPyMT) mouse. C57 refers to the C57BL/6JN1cr mouse.

FIG. 2A shows a female mitochondrial-nuclear exchanged mouse [C57 mtDNA/FVB/N-TgN(MMTVPyMT) nuclear DNA (nDNA)] at 3 months of age.

FIGS. 2B and 2C show mtDNA haplotyping gels (Asp I and Bcl I, respectively) to confirm mtDNA haplotype from ear clip DNA; lane 1 is the mouse in FIG. 2A.

FIGS. 3A, 3B and 3C are graphs showing isometric tension measured in aortic segments from 12-week old male C57 and C3H mice. Thoracic aorta were cut into 2 mm ring segments and suspended from a force-displacement transducer in an isolated tissue bath. To assess endothelium-dependent relaxation, indomethacin-treated rings were contracted with phenylephrine (PE) (FIG. 3A) followed by addition of acetylcholine (Ach) (FIG. 3B). Sodium nitroprusside (SNP) was added to contracted rings to determine endothelial independent relaxation (FIG. 3C). Relaxation was quantified as percent decrease in vessel tension of the pre-existing tone generated by PE. Data are the mean from 3-4 ring segments from each animal (N=4 mice/group). Asterisks (*) indicate significant difference (P<0.05) between the two mouse strains.

FIG. 4A shows mice generated from mitochondrial-nuclear exchange. Coat color indicates nDNA genome (C3H—brown, C57—black), which has been confirmed via SNP analysis of 38 markers (Jackson Labs, Bar Harbor, Me.). Numbers on mice indicate mtDNA RFLP analyses in FIGS. 4B and 4C. Number 3, brown coat, is the mouse with nDNA from C3H and mtDNA from C57 mice.

FIG. 4B shows AspI RFLP analysis verifying mtDNA genotype. PCR products from C57 mtDNAs were cleaved by AspI to yield 274 bp and 111 bp fragments, whereas C3H mtDNAs were uncut (385 bp).

FIG. 4C shows BclI RFLP analysis verifying mtDNA genotype. PCR products from C57 mtDNAs were uncut (204 bp) whereas C3H mtDNAs were cleaved by BclI to yield 166 bp and 38 bp fragments.

FIG. 5 is a schematic summarizing the process of creating mitochondrial-nuclear exchanged animals.

FIG. 6A shows polarographic trace of isolated heart mitochondria. State 3 respiration is initiated by addition substrate (glutamate+malate) and ADP (125 nmoles) and total O2 consumption determined until the return to state 4 respiration (occurs when all the ADP is consumed by phosphorylation to ATP). Addition of oligomycin abolishes ADP-induced respiratory stimulation which is subsequently relieved by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP).

FIG. 6B is a graph showing the ADP/O ratio of C57BL/6J (C57) and C3H/HeN (C3H) mice relative to C57BL/6J control mitochondria. The ADP/O ratio is calculated as the ratio of nanomoles of ADP added, to nanoatoms of oxygen consumed during state 3 respiration. Data are expressed relative to the C57BL/6J control mitochondria because results were obtained from 5 independent experiments, with C57BL/6J mitochondria serving as the control for each separate experiment (N=2 per strain per experiment). The ADP/O mean+SE over all experiments was C57−2.88+0.05; C3H−2.39+0.04). Asterisk (*) indicates a significant difference (P<0.05) exists from C3H/HeN mouse mitochondria.

FIG. 7 shows RFLP analysis of mtDNA COIII subunit. Specifically, a gel of AspI restriction digest of 385 bp PCR products from C57 and C3H mice is shown. The C57 mtDNA was cleaved into 274 bp and 111 bp, whereas the C3H mtDNA was not.

FIGS. 8A and 8B show cytochrome c oxidase activities in C57 and C3H mice. Mitochondria were prepared from whole aortas harvested from 10-week old male C57BL6 and C3H/HeN mice. In FIG. 8A, complex IV activity was determined by measuring the oxidation of reduced cytochrome c at 550 nm (37° C.) in isolated mitochondria. Asterisks (*) indicate a significant difference exists between C57 and C3H mitochondria. In FIG. 8B, immunoblot analysis showing the relative level of complex IV subunit II from an aliquot of the samples used for complex IV activity.

FIG. 9 shows Respiratory Control Ratios (RCR) in C57 and C3H mouse hearts. Mitochondria were isolated from age-matched (10 week old) C57and C3H mouse hearts, and oxygen consumption determined in the presence of an electron donor (glutamate/malate) with and without ADP (state 3 and 4 respiration, respectively). The bars represent the respiratory control ratio (RCR, equal to state 3 respiration rate divided by state 4 respiration rate); C3H hearts had significantly lower RCR values, indicating that oxygen utilization (in terms of generating ATP) by the mitochondrion is less efficient in the C3H hearts. N=3/group.

FIG. 10 is a bar graph showing mitochondrial membrane potentials in C57 and C3H mice. Mitochondria were prepared from hearts harvested from 10-week old male C57BL6 and C3H/HeN mice (fed chow diets) and JC-1 fluorescence determined at 535 nm and 590 nm emissions on a CytoFluor 4000 fluorometer. Asterisks (*) indicate that a significant difference exists between C57 and C3H mitochondria.

FIG. 11 shows the effect of diet on mtDNA damage in aortas from C57 and C3H mice. Male C57 and C3H mice were fed either chow or high fat diets (4% or 21% fat, respectively) from 6-10 weeks of age. DNA was extracted from aortas and QPCR was performed to quantify mtDNA damage. Asterisks (* or **) indicate significant differences (P<0.05) exist from chow fed C57 or high fat C3H, respectively. N=3 per group.

FIG. 12 shows relative levels of Amplex Red fluorescence from mitochondria isolated from C57 and C3H mouse aortas. 10-week old male C57 and C3H mice were sacrificed, and mitochondria isolated from aortas (2 aortas were pooled/sample). 50 μg of mitochondrial protein were incubated with glutamate/malate, ADP, Amplex Red, and HRP. Relative fluorescence indicates increased oxidant production in C57 mitochondria relative to C3H. Asterisks (*) indicate significant differences (P<0.05) exist. N=6 mice, or 3 samples (C57); N=6 mice, or 3 samples (C3H).

FIG. 13 shows isometric tension measured in aortic segments from 12-week old male C57 and C3H mice. Briefly, thoracic aorta were cut into 2 mm ring segments and suspended from a force-displacement transducer in an isolated tissue bath. To assess endothelium-dependent, NO-mediated relaxation, indomethacin-treated rings were contracted with phenylephrine followed by addition of acetylcholine. Relaxation was 20674-0101001 quantified as percent decrease in vessel tension of the pre-existing tone generated by phenylephrine. Data are the mean from 3-4 ring segments from each animal (N=4 mice/group). Asterisks (*) indicate significant difference (P<0.05) between the two mouse strains.

FIGS. 14A, 14B and 14C are graphs showing isometric tension measured in aortic segments from 14-week old male C57mtDNA/C57nDNA; C3HmtDNA/C3HnDNA; C3HmDNA/C57nDNA; and C57mtDNA/C3HnDNA mice. To assess endothelium-dependent, NO-mediated relaxation, indomethacin-treated rings were contracted with phenylephrine (PE) (FIG. 14A) followed by addition of acetylcholine (Ach) (FIG. 14B). Sodium nitroprusside (SNP) was added to contracted rings to determine endothelial independent relaxation (FIG. 14C). Relaxation was quantified as percent decrease in vessel tension of the pre-existing tone generated by PE. Data are the mean from 3-4 ring segments from each animal (N=3 mice/group). Asterisks (* and **) indicate significant difference (P<0.05) between the C57mtDNA/C57nDNA and C57mtDNA/C3HnDNA, respectively.

FIG. 15 shows mtDNA phylogeny derived from known mtDNA sequences from mice (GenBank) also known to have differential susceptibility to cardiovascular disease (CVD). CVD susceptibility is characterized as “Very Resistant,” “Resistant,” and “Susceptible” based upon review of the literature; “very resistant” indicates no atherosclerotic lesion formation, “resistant” indicates modest lesion formation relative to the “susceptible” C57 mouse. Amino acid or tRNA changes are indicated next to the location of each mtDNA mutation.

DETAILED DESCRIPTION

MtDNA mutations have been linked with human disease. For example, studies have shown that specific mtDNA mutations and haplotypes are associated with increased risk for diseases thought or known to have an environmental component in humans (e.g., deafness, blindness, Alzheimer's disease, diabetes and cancer). Similarly, studies have shown that the mtDNA haplotype can influence tumor growth and age-related deafness in mice. Mitochondrial haplotypes thought to be associated with tightly coupled mitochondria appear to be more prone to certain types of cancer and neurodegenerative diseases associated with oxidative stress and/or somatic mutation. Because it is difficult to examine multiple molecular mechanisms related to many of the aforementioned aspects of mitochondria (e.g., damage, membrane potential, oxidant stress, respiratory activity and enzyme function) in human populations, animal models are required to test the molecular aspects of the mitochondrial-nuclear interaction in determining individual disease susceptibility.

Thus, provided herein are chimeric animals, or cells, tissues or organs thereof with the mitochondrial DNA (mtDNA) from of one cell type (genetic background) and the nuclear DNA (NDNA) from of another cell type, also referred to herein as mitochondrial-nuclear exchanged animals, cells, tissues or organs. Within the cell, mitochondria and the nucleus interact in a manner that ultimately determines how the cell will function and respond to various endogenous and exogenous factors. Consequently, how mitochondria and the nucleus interact determines an individual's susceptibility to disease and the individual's ability to adapt to changes in the environment. Hence, the provided methods allow exchange of mitochondrial and nuclear genetic materials (i.e., putting a cell nucleus into a cytoplasm containing mitochondria that are typically not associated with that nuclear DNA) and model systems for determining how mtDNA impacts disease development and susceptibility to factors associated with disease risk and adaptation to general environmental changes. The provided chimeric models and cells are used to determine the mechanistic and genetic basis of disease susceptibility and resistance (e.g., cardiovascular disease, cancer, diabetes, musculoskeletal, neurological, obesity, aging, fitness, and the like).

FIG. 5 summarizes the process of creating mitochondrial-nuclear exchanged animals. Specifically, pro-nuclear embryos are harvested from genetically distinct donors (in terms of mitochondrial and nuclear DNAs), enucleated, and the nucleus from one donor is introduced into the enucleated cell of the other, generating an re-nucleated embryo with the nuclear DNA from one strain (e.g., strain B) and the mitochondrial DNAs (e.g., strain A) from the other. These embryos are implanted into surrogate mothers who carry the transgenic embryo to term. The genetic identity of the progeny is confirmed via genotyping tail and ear clips from the offspring. Females of desired mtDNA-nDNA genotype are then used as founding breeders to establish colonies useful as model systems of mitochondrial-nuclear interaction and disease susceptibility (e.g., females from these colonies are used to establish mitochondrial-nuclear exchanged colonies of any transgenic animal currently available for biomedical research).

Provided are animals, cells, tissues or organs comprising mitochondrial DNA (mtDNA) from a subject susceptible to a disease or disorder and nuclear DNA (nDNA) from a subject that is not susceptible to the disease or disorder. Optionally, the nDNA is from a wild-type subject. Optionally, the nDNA is from a subject resistant to the disease or disorder.

As used herein, a subject susceptible to a disease or disorder refers to a subject having or at risk for developing one or more symptoms associated with the disease or disorder. Thus, subjects susceptible to the disease or disorder have an increased rate of occurrence or a faster onset of one or more symptoms of the disease or disorder as compared to a wild-type subject. Subjects susceptible to the disease or disorder may have a family history or known genetic predisposition for developing the disease or disorder. As used herein, wild-type subjects refer to subjects without the disease or disorder and without enhanced resistance or susceptibility to a disease or disorder of interest. As used herein, a subject resistant to the disease or disorder refers to a subject less likely to develop the disease or disorder than a wild-type subject. Thus, subjects resistant to the disease or disorder have a decreased rate of occurrence or a lower onset of the disease or disorder as compared to a wild-type subject. Subjects resistant to the disease or disorder can include partial or total resistance as compared to wild-type subject. Further a subject can be referred to as wild-type in the context of one disease or disorder while referred to as susceptible in the context of another disease or disorder. For example, a subject can be referred to as wild-type in the context of cancer, but referred to as susceptible in the context of cardiovascular disease. In other words, the subject is not prone to cancer but is prone to cardiovascular disease.

Also provided are animals, cells, tissues or organs comprising mitochondrial DNA (mtDNA) from a subject resistant to a disease or disorder and nuclear DNA (nDNA) from a wild-type subject or a subject that is susceptible to the disease or disorder.

The disease or disorder is, for example, cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder. Optionally, the cell is an oocyte or an embryonic cell. Also provided are zygotes and embryos, such as pro-nuclear embryos comprising the cells.

Optionally, the cells are obtained from any animal including, mammals, birds and amphibians. Suitable mammalian sources include sheep, bovines, ovines, pigs, horses, rabbits, guinea pigs, mice, hamsters, rats, primates, and the like. Optionally, the pro-nuclear embryos are obtained from mice. Optionally, the cells are not human cells.

Other human and animal cells useful in the present disclosure include, by way of example, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, and other muscle cells. Moreover, the human cells used for nuclear exchange may be obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, and the like. These are just examples of suitable donor and recipient cells. Suitable donor and recipient cells may be obtained from any cell or organ of the body. This includes all somatic or germ cells.

Methods of screening for agents useful for treating a disease or disorder are provided. Such a screening method comprises the steps of providing a mitochondrial-nuclear exchanged cell, tissue or organ, contacting the cell, tissue or organ with a candidate agent to be tested and determining whether the agent prevents or reduces one or more symptoms of the disease or disorder. Optionally, the cells, tissues or organs are obtained from a mitochondrial-nuclear exchanged animal. Suitable tissues and organs include, skin, lung, muscle, cartilage, bone, bone marrow, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urinary organs, and the like.

By way of example, cardiovascular disease is associated with increased levels of oxidants, and an agent useful in treating cardiovascular disease causes decreased levels of oxidants. Thus, the determining step is, optionally, determining the level of oxidants in the cells. Alternatively, the determining step includes assessing mitochondrial function in the cells to determine whether the agent is useful in treating the disease or disorder. Such methods are known and are described in the Examples below. By way of another example, cancer is associated with increased cellular proliferation as compared to wild-type cells. Thus, the determining step is, optionally, determining the rate of proliferation of the cells.

Such methods allow one skilled in the art to select candidate agents that are useful in treating, reducing or preventing one or more symptoms of the disease or disorder. Such agents may be useful as active ingredients included in pharmaceutical compositions for treating a subject suffering from the disease or disorder.

Also provided are chimeric animals comprising a plurality of the provided cells. Such chimeric animals are also referred to herein as mitochondrial-nuclear exchanged animals. Thus, provided herein are chimeric animals comprising mitochondrial DNA (mtDNA) from a subject susceptible to a disease or disorder and nuclear DNA (nDNA) from a subject that is not susceptible to the disease or disorder. Also provided herein are chimeric animals comprising mtDNA from a subject resistant to a disease or disorder and nDNA from a wild-type subject or a subject that is susceptible to the disease or disorder. By way of example, the animal is a mouse comprising mtDNA from C57BL/6J mice and nDNA from C3H/HeN mice. Optionally, the animal is a mouse comprising mtDNA from C3H/HeN mice and nDNA from C57BL/6J mice. By way of another example, the animal is a mouse comprising mtDNA from NZB/B1NJ mice, C57BL/6J mice or AKR/J mice and nDNA from FVB/N-TgN(MMTVPyMT) mice. By way of another example, the animal is a mouse comprising mtDNA from NZB/B1NJ mice and nDNA from C57BL/6J mice. Optionally, the animal is a mouse comprising mtDNA from C57BL/6J mice and nDNA from NZB/B1NJ mice.

As used herein, the term chimeric animal refers to non-human animals, including, mammals, amphibians and birds. Specifically, examples include sheep, feline, bovines, ovines, pigs, horses, rabbits, guinea pigs, mice, hamsters, rats, primates, and the like. Optionally, the animal further comprises a mutation in a gene associated with the disease or disorder. Optionally, the gene is not expressed or the protein expressed by the gene is non-functional. For example, the animal optionally comprises a mutation in SOD2 or ApoE such that SOD2 or ApoE is not expressed or the protein expressed by the gene is non-functional.

Also provided are progeny of the chimeric animals and progeny animals resulting from a cross between the female chimeric animal and a knockout mouse, wherein the knockout mouse comprises a mutation in at least one gene associated with the disease or disorder such that the gene is not expressed or the protein expressed by the gene is not functional. Optionally, the knockout mouse is susceptible to the disease or disorder or resistant to the disease or disorder.

Also provided are methods for producing a mitochondrial-nuclear exchanged animal and a mitochondrial-nuclear exchanged animal made by the provided method. The method comprises selecting an animal susceptible to a disease or disorder or an animal resistant to the disease or disorder; selecting an animal that is not susceptible to the disease or disorder or an animal that is not resistant to the disease or disorder; harvesting the pro-nuclear embryos from each of the animals; enucleating the embryos; transferring the nucleus from the embryo of the animal that is not susceptible to the disease or disorder to the enucleated embryo of the animal susceptible to the disease or disorder to make a resulting embryo, wherein the resulting embryo has mtDNA from the animal susceptible to the disease or disorder and the nDNA from the animal that is not susceptible to the disease or disorder; and transferring the resulting embryo into an appropriate host. The transferred embryo is then allowed to develop into a progeny animal. The progeny animal is a mitochondrial-nuclear exchanged animal. Optionally, the animal that is not susceptible to the disease or disorder is from a wild-type animal or an animal resistant to the disease or disorder. Optionally, the method further comprises selecting female mitochondrial-nuclear exchanged animals for further breeding.

A method of generating progeny of the female mitochondrial-nuclear exchanged animals is provided by crossing the females with a knockout animal, wherein the knockout animal comprises a mutation in at least one gene associated with the disease or disorder such that the gene is not expressed or the protein expressed by the gene is not functional. Optionally, the method further comprises selecting progeny animals of the cross that comprise mtDNA from the mitochondrial-nuclear exchanged animals and nDNA from the knockout mouse.

In the provided methods, the disease or disorder of interest is, optionally, cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, or musculoskeletal disorder. Other disease and disorders are contemplated and can be of interest when animals with reduced resistance or enhanced susceptibility are accessible.

Methods of nuclear exchange are known and include the methods described in the examples below and those described in, for example, U.S. Publication No. 2003/0032180; U.S. Publication No. 2005/0120402; U.S. Publication No. 2005/0095704; and U.S. Patent No. 6,603,059, which are incorporated by reference herein in their entireties. Nuclear exchange is also described in U.S. Pat. Nos. 4,944,384; 5,057,420; Campbell et al., Theriogenology, 43:181 (1995); Collas et al., Mol. Report Dev., 38:264-267 (1994); Keefer et al., Biol. Reprod., 50:935-939 (1994); Sims et al., Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, which are incorporated by reference in their entireties herein.

Animals known to be susceptible or resistant to specific diseases are obtained, for example, from the Jackson Laboratory or other commercial or non-commercial sources. By way of example, mice used in the provided methods include, but are not limited to, C57BL/6J, C3H/HeN, AKR/J, FVB strains, BALB/c strains, A/J strains, 129 strains and DBA/2 strains. Wild-type animal models can also be obtained from commercial and non-commercial sources. Mouse models known to be susceptible to specific diseases are listed in the Mouse Phenome Database at http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home, which is incorporated herein by reference in its entirety. For example, 29 strains are listed in the Mouse Phenome Database as being susceptible to cancer. By way of another example, over 50 strains are listed in the Mouse Phenome Database as being susceptible to cardiovascular disease. A few mouse models are discussed in detail below; however, any subject susceptible, resistant or wild-type can be used in the provided methods to generate mitochondrial-nuclear exchanged animals.

Mice useful in the provided methods include, A/J mice, which are susceptible to cancers and resistant to cardiovascular disease. A/J mice have a high incidence of spontaneous lung adenomas, lung tumors that readily develop in response to carcinogens, and mammary adenocarcinomas. A/J mice fed an atherogenic diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) fail to develop atherosclerotic aortic lesions in contrast to several highly susceptible strains of mice. In addition to atherosclerosis resistance, A/J mice are resistant to diabetes, obesity, insulin resistance and glucose intolerance.

FVB/N-TgN(MMTV-PyMT) mice carrying the (MMTV-PyVT) transgene are susceptible to cancer.

C57BL/6 mice are commonly used as a general purpose strain and background strain. This strain is refractory to many tumors. C57BL/6J mice are also commonly used in the production of transgenic mice. This strain can be used as a wild-type strain in many contexts. However, the C57BL/6J strain can be used in the context of cardiovascular disease as a susceptible animal since C57BL/6J mice are highly susceptibility to diet-induced obesity, type 2 diabetes, and atherosclerosis. Thus, C57BL/6J mice can be considered a subject susceptible to obesity, diabetes or cardiovascular disease or as a wild-type subject in the study of other diseases such as immunological diseases or cancer.

NZB/B1NJ mice display a number of autoimmune abnormalities including hemolytic anemia, elevated levels of immunoglobulin, anti-DNA antibodies, anti-thymocyte antibodies, and circulating immune complexes causing glomerulonephritis. F1 hybrids of NZB/B1NJ and NZW/LacJ (NZBWF1/J) are used as a model for autoimmune disease resembling human systemic lupus erythematosus. NZB/B1NJ mice, fed an atherogenic diet (1.25% cholesterol, 0.5% cholic acid and 15% fat), fail to develop atherosclerotic aortic lesions. NZB/B1NJ mice can be used as animals susceptible to autoimmune disorders or as animals resistant to cardiovascular disease. By way of example, mice known to be susceptible (NZB/B1NJ) to automimmune disorders will be used in nuclear exchange experiments to generate mice with a NZB/B1NJ mtDNA haplotype and a nuclear genome of a normal mouse or a mouse resistant to autoimmune disorders. These mice are generated using the method set forth in Example 1 below.

AKR/J mice are widely used in cancer research for their high leukemia incidence. AKR/J mice, however, are relatively resistant to aortic lesion formation on a semi-synthetic high fat diet and are hyporesponsive to diets containing high levels of fat and cholesterol. Thus, AKR/J mice are referred to as resistant to cardiovascular disease, but susceptible to cancer, and can be used accordingly in the present methods and cells.

DBA/2J is a widely used strain in a large number of research areas, including cardiovascular biology, neurobiology, and sensorineural research. DBA/2J mice show a low susceptibility to developing atherosclerotic aortic lesions (20 to 350 μm2 atherosclerotic aortic lesions/aortic cross-section) following 14 weeks on an atherogenic diet (1.25% cholesterol, 0.5% cholic acid and 15% fat). They also exhibit high-frequency hearing loss beginning roughly at the time of weaning/adolescence (between 3-4 weeks of age) and becoming severe by 2-3 months of age. DBA/2J mice also show an extreme intolerance to alcohol and morphine. Thus, DBA/2J mice can be used as susceptible to neurologic disorders and hearing loss, but resistant to cardiovascular disease and substance abuse or addiction.

Methods of screening for agents useful for treating a disease or disorder are provided comprising the steps of providing a mitochondrial-nuclear exchanged animal comprising mtDNA from an animal susceptible to the disease or disorder and nDNA from an animal not susceptible to the disease or disorder, administering to the animal an agent to be tested, and determining whether the agent prevents or reduces one or more symptoms of the disease or disorder. Optionally, the animal not susceptible to the disease or disorder is a wild-type animal or an animal resistant to the disease or disorder. Optionally, the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, diabetes, neurological disorder, aging, metabolic disorder, immune disorder, obesity, and musculoskeletal disorder. Optionally, the disease is cancer and the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from a mouse selected from the group consisting of FVB/N-TgN(MMTVPyMT), AKR/J, and A/J mice. Optionally, the disease is cardiovascular disease and the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from C57BL/6J or DBA/2J mice. Optionally, the mitochondrial-nuclear exchanged animal is a mouse comprising mtDNA from C57BL/6J mice and NDNA from C3H/HeN mice. Optionally, the mitochondrial-nuclear exchanged animal is a mouse comprising nDNA from a mouse selected from the group consisting of C57BL/6J, 129, A/J, BALB/c and C3H/HeN mice.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a chimeric animal is disclosed and discussed and a number of modifications that can be made to the chimeric animal are discussed, each and every combination and permutation of the chimeric animal, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of using the disclosed compositions or animals. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

A number of aspects have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, other aspects are within the scope of the claims.

Examples

Example 1

Mitochondrial Influence on Breast Cancer Metastasis Susceptibility

It is widely appreciated that carcinogenic risk may be influenced by a combination of genetic and environmental factors that influence an individual's predilection to metastatic tumor formation. In this respect, numerous studies have investigated and reported that nuclear genetic differences may influence susceptibility to breast cancer; other reports suggest a potential role for the mitochondrion in influencing tumor metastatic potential. In this regard, an important consideration currently lacking in determining individual susceptibility to breast cancer metastasis is the potential role for mitochondrial-nuclear interaction.

Mitochondria have been implicated to play a significant role in the etiology of a variety of diseases, including cancer. Certain human mitochondrial DNA haplotypes appear to increase the risk for certain types of cancer and tumor growth in mice. Genetic background also influences metastatic mammary tumor formation in mice. To test whether mitochondrial-nuclear exchanged interaction influences metastatic susceptability, a molecular genetic approach was developed to investigate the role of mitochondrial genetic background on breast cancer metastasis. Specifically, mice known to be resistant (C57BL/6JN1cr) to metastasis in a metastatic mammary tumor model (FVB/N-TgN(MMTVPyMT transgenic mouse) were used in nuclear exchange experiments to generate mice with a C57BL/6J mtDNA haplotype and FVB/N-TgN nuclear genome and vice versa.

Specifically, fertilized oocytes were collected from C57BL/6JN1cr, and FVB/N-TgN(MMTVPyMT superovulated donor females. Enucleation of metaphase II oocytes and donor cell nuclear injections were conducted. Briefly, PMSG and hCG were administered to 4-6 wk old donor females, placed with stud males, and oocytes collected at 0.5 days post coitum (dpc). After cumulus cell removal, oocytes (15-20) were placed in a micromanipulation chamber containing M2 medium supplemented with cytoskeletal inhibitors cytochalasin B(5 ug/mL) and Colcemid(0.1 ug/mL) for 5-10 minutes. Using a holding pipette to immobilize an oocyte, the zona pellucida is then “cored” using a single low duration high-intensity piezo-pulse (Primetech piezoelectric drill) from a ground 45 degree-angle enucleation pipette, both pronuclei were carefully aspirated and expelled. When all oocytes had been enucleated, donor oocytes (those providing new nuclear material) were placed in the micromanipulation chamber, and pronuclei were again aspirated and immediately injected into the previously enucleated (recipient) oocytes. Reconstructed nuclear transfer embryos were then rinsed in fresh M2 media and electrofused in 25 uL droplets under oil. Reconstructed nuclear transfer embryos were immediately implanted into the oviducts of 0.5 dpc pseudopregnant females or cultured overnight to the 2-cell stage and then transferred. Surrogate mothers underwent C-sections if pups were not born naturally on their due date and fostered to ICR foster mothers.

FIG. 1 shows the number of mitochondrial-nuclear exchanges performed resulting in term births, and surviving animals (6 weeks of age). Labels on the X-axis indicate mtDNA haplotype/nuclear genotype. PyMT refers to the FVB/N-TgN(MMTVPyMT) mouse. C57 refers to the C57BL/6JN1cr mouse.

FIG. 2A shows a female mitochondrial-nuclear exchanged mouse [C57 mtDNA/FVB/N-TgN(MMTVPyMT) nuclear DNA (nDNA)] at 3 months of age. FIGS. 2B and 2C show mtDNA haplotyping gels (Asp I and Bcl I, respectively) to confirm mtDNA haplotype from ear clip DNA; lane 1 is the mouse in panel A.

These data shown mitochondrial-nuclear exchange techniques can be used to successfully generate FVB/N-TgN(MMTVPyMT) transgenic mice on different mtDNA haplotype backgrounds, providing a novel means for direct assessment of mitochondrial-nuclear role(s) on cancer metastasis. As an example of the feasibility of using the mitochondrial-nuclear exchange (MNX) mouse model, one female and three male MNX mice were generated with a C57BL/6J mtDNA and a PyMT nuclear FVB/N genome (mtDNAC57BL/6::nDNAPyMT) (Table 1). Because F1 progeny from PyMT ♂ X C57BL/6 ♀ have a suppressed metastasis index, it was thought that MNX mtDNAC57BL/6::nDNAPyMT mice would have suppressed tumor formation and metastatic potential compared to mice harboring the FVB/N mtDNA (the “wild-type” mtDNA for the PyMT transgenic) and PyMT nuclear genome (mtDNAFVB/N::nDNAPyMT).

TABLE 1
A summary of the study from the first four
mtDNAC57BL/6::nDNAPyMT mice produced.
Onset of PrimaryNumber of Lung
GenderAgeBreast TumorMetastases
MNX Femalea110 days at sacrifice 79 days3
MNX Malea238 days at sacrifice107 daysNone detected
MNX MaleaCurrenlty 308 days, notNone detectedND
yet sacrificed
MNX MaleaCurrently 308 days, notNone detectedND
yet sacrificed
Female controlsb94 ± 2.35 days at67.22 ± 3.07 days24.63 ± 5.39
sacrifice
Male controlsb,cAverage age at 83 ± 20 daysSpecific number not
sacrifice not providedreported, multiple,
80% penetrance
ND—not determined
amtDNAC57B1/6::nDNAPyMT mice
bmtDNAFVB/N::nDNAPyMT mice; Control data are listed as mean + SEM
cMale control data are from Hazen and Heinecke, J. Clin. Invest. 99: 2075-2081 (1997).

“Wild-type” (mtDNAFVB/N::nDNAPyMT) mice develop mammary tumors and metastases with 100% penetrance within 70 days and 90 days, respectively. The MNX female mouse (mtDNAC57BL/6::nDNAPyMT) exhibited slightly longer latency (79 days), but significantly fewer surface lung metastases (3 versus 25 for wild-type mice). Of the three male MNX mice (mtDNAC57BL/6::nDNAPyMT) generated, only one has developed a mammary tumor (at 107 days), which is substantially longer than “wild-type” PyMT males (mean latency 83 days). When euthanized at 238 days, no metastases were found. The other two males were alive and tumor-free at 320+ days.

These data show that pre-existent normal mitochondrial haplotypes or polymorphisms influence breast cancer latency and metastatic efficiency.

Example 2

Examples of Mitochondrial-Nuclear Exchanged Mice as Cancer Animal Models

Mice known to be susceptible (AKR/J) or resistant (NZB/B1NJ) to metastasis in a metastatic mammary tumor model (FVB/N-TgN(MMTVPyMT transgenic mouse) are used in nuclear exchange experiments to generate mice with a NZB/B1NJ mtDNA haplotype and FVB/N-TgN nuclear genome, and a mouse with a AKR/J mtDNA and FVB/N-TgN nuclear genome. These mice are assessed for metastatic tumor formation compared to mice with FVB mtDNA and FVB/N-TgN nuclear genomes. These mice are generated using the method set forth in Example 1.

Example 3

Mitochondrial-Nuclear Exchanged Mice and Cardiovascular Disease Susceptibility

As described herein, mitochondrial-nuclear exchange successfully generated mice with mtDNAs of one strain and the nDNA of another. C57BL/6J mice are prone to cardiovascular disease while C3H/HeN mice are not. C3H mice have increased sensitivity to endothelial dependent vasorelaxation compared to C57 mice. Because C57 mice are more susceptible to atherogenesis compared to C3H mice, it was hypothesized that endothelial dependent vasorelaxation would be decreased in C57 mice relative to C3H mice. Consequently, vessel dilatation studies were performed on C57BL/6 and C3H/HeN mouse aortas harvested from 12 week old mice. FIGS. 3A, 3B and 3C reveal that C3H mice were more sensitive to acetylcholine induced relaxation, whereas no differences were observed in endothelial independent relaxation (SNP), consistent with the hypothesis that C57 mice have decreased endothelial dependent vessel relaxation compared to C3H animals.

To test the hypothesis that the noted differences in mouse strain susceptibility to CVD are related to mtDNA haplotype, mice were generated that have the mtDNA of a susceptible strain (C57) and the NDNA of a resistant strain (C3H) and vice versa. FIG. 4A shows 6 female mice generated by mitochondrial-nuclear exchange using C57 and C3H pronuclear embryos. The 5 mice with black coats have C57 black nuclear DNA, as confirmed by both coat color and typing of 38 strain specific SNPs. These mice also have C3H mouse mitochondrial DNAs (FIGS. 4B and 4C: lanes 1,2 and 4-6), as determined by Asp I and Bcl I RFLP analyses (FIG. 4B and 4C, respectively). The mouse with brown coat color (#3) has C3H nuclear DNA (confirmed by SNP analysis) and a C57 mtDNA (FIG. 4B and 4C: Lane 3) by RFLP analysis. These results confirm the feasibility of the proposed mitochondrial-nuclear exchange experiments.

Example 4

Mitochondrial Influence on Cardiovascular Disease Susceptibility

Because it is known that certain mouse strains have differential susceptibilities to CVD development, mitochondrial function and genetics may be important factors in influencing individual CVD susceptibility differences. Such differences in mice known to be susceptible or resistant to CVD were determined. C57BL/6J mice, referred to in Example 4 as C57 mice, are susceptible to dietary induced atherogenesis, whereas C3H/HeN, referred to in Example 4 as C3H mice, are not.

Experiments were performed to distinguish between the two mtDNA haplotypes of C57 and C3H. In this respect, C57 and C3H differed at nt 9348 within the COIII gene (G to A, resulting in the change of a highly conserved Val248 to Ile248 in C57 to C3H, respectively). This change abolishes a AspI site in the C3H mtDNA. FIG. 7 shows a 385 bp PCR product from C57 and C3H mice digested with AspI restriction enzyme, indicating that the C57 and C3H mtDNAs are distinguishable. Val248 is conserved between C57 mice, rats, gorillas, humans, frogs and trout.

To determine whether the missense mutation in cytochrome oxidase subunit III conveyed any functional effects upon electron transport, mitochondria were isolated from C3H and C57 mice (fed chow diets) and assessed for complex IV activity. FIG. 8A shows that C3H mitochondria had significantly decreased complex IV activity relative to C57 mitochondria. Immunoblot analyses (FIG. 8B) from aliquots used in the enzyme analysis revealed no significant differences in subunit II of complex IV, showing that the observed differences seen in the enzymatic activity were not due to differences in complex IV amounts. These findings were consistent with the concept that there will be functional differences between the atherogenic susceptible C57 and resistant C3H mice.

To determine whether the mitochondria from C3H mice were different from C57 in terms of oxygen utilization and ATP generation, respiratory control ratios (RCR =state 3/state 4 respiration; oxygen consumption rates in the presence and absence of ADP, respectively) were quantified from mitochondria isolated from heart tissues harvested from male C3H and C57 mice on chow diets. FIG. 9 shows that the RCR in C3H mice appeared decreased relative to the C57 mice, showing that mitochondrial oxidative phosphorylation was less “coupled” to oxygen consumption in the C3H mice, consistent with the hypothesis that C3H have less coupled mitochondria relative to C57 mice, and therefore, are resistant to the oxidant stress associated with CVD risk factors.

To evaluate the relative efficiencies of mitochondrial ATP generation coupled to oxygen consumption in C57BL/6J and C3H/HeN mice, 10−C57BL/6J and 10−C3H/HeN mice were harvested at 14 weeks of age over a series of 5 days (N=2 C57BL/6J, and 2−C3H/HeN mice per experiment, per day), and a known amount of ADP (125 nmoles) was added to equal amounts of heart mitochondria isolated from C57 or C3H mice to induce state 3 respiration (malate/glutamate+ADP). The amount of oxygen consumed was quantified to the point of return to state 4 respiration (occurs when all the ADP is consumed by phosphorylation to ATP: FIG. 6A). The ADP/O ratio relative to the C57BL/6J mouse mitochondrial control (determined from 5 independent experiments; 2−C57BL/6J and 2−C3H/HeN mice per experiment), is presented in FIG. 6B, showing that C3H/HeN mice have significantly lower ADP/O ratios compared to C57BL/6J mice. Low ADP/O ratios reflect decreased efficiency of oxygen utilization to make ATP and, thus, less efficient mitochondria. These results showed that C3H/HeN mitochondria consumed more oxygen compared to C57BL/6J mitochondria, per ADP molecule phosphorylated, consistent with the hypothesis that C3H/HeN mice are less energetically efficient compared to C57BL/6J mice.

Because the data above suggested that differences existed between the mitochondria from C3H and C57 mice, JC-1 fluorescence was utilized to determine mitochondrial membrane potentials. JC-1 exists as a green fluorescent monomer at low concentrations or low membrane potential, whereas at higher concentrations (>0.1 μm) it forms a red-fluorescent “J-aggregate.” Consequently, the ratio of red-to-green JC-1 fluorescence is dependent only on the mitochondrial membrane potential and not on other factors that may influence single-component fluorescence signals, such as mitochondrial size, shape and density. FIG. 10 shows that mitochondria isolated from C57 mouse hearts had significantly higher membrane potentials than age matched (10-week old) C3H mitochondria. These findings are consistent with the hypothesis that C3H mice have mitochondria that are less coupled than those of C57 mice.

Because the data above were consistent with the hypothesis that C3H mice were less coupled relative to C57 mice, the impact of a high fat diet on mtDNA damage was assessed in aortic tissues from both C57 and C3H mice. Male C3H and C57 mice were fed either a chow or high fat diet from 6 to 10 weeks of age, and mtDNA damage was determined from aortas. FIG. 11 shows that a high fat diet significantly increased mtDNA damage in C57 mice, whereas damage was not significantly increased in the C3H mouse.

To determine whether differences in oxidant levels existed between mitochondria from C57 and C3H mice, isolated mitochondria were assessed for oxidant generation by Amplex Red fluorescence (in the presence of HRP, reacts with H2O2). FIG. 12 shows that C57 mitochondria exhibited significantly higher levels of fluorescence compared to C3H mitochondria, consistent with increased oxidant production.

Increased levels of SOD2 or uncoupling proteins (UCPs) could explain, in part, the observed differences between C3H and C57 mitochondria. To determine whether differences in SOD2 protein or enzymatic activity or UCP levels existed, SOD2 activity and protein levels were assessed, as were UCP 2 and 3 transcript levels (RT-PCR) from aortas and hearts from 10-week old C57 and C3H mice. No differences were found, suggesting that the differences between C57 and C3H mice were not due to changes in SOD2 or UCP 2 or 3 levels.

Because data suggested that C57 mitochondria were more tightly coupled than C3H, and that higher oxidant production (presumably O2−) appeared to be associated with C57 mitochondria, it was hypothesized that endothelial dependent vasorelaxation would be inhibited in C57 mice relative to C3H mice (due to increased oxidant stress). FIG. 13 shows that C3H mice were more sensitive to acetylcholine induced vasorelaxation, consistent with the principal that these animals had greater NO bioavailability by virtue of decreased mitochondrial oxidant production.

FIGS. 14A, 14B and 14C show that vascular function segregates with mitochondrial haplotype in mitochondrial-nuclear exchange mice. To evaluate the impact the mitochondrial genetic background on vascular function, 14 week old mitochondrial-nuclear exchange male C3HmtDNA/C57nDNA and C57mtDNA/C3HnDNA mice were used in vessel relaxation studies and compared to age-matched wild-type C57BL/6 (C57mtDNA/C57nDNA) and C3H/HeN (C3HmtDNA/C3HnDNA) male mice. FIGS. 14A, 14B and 14C show that while all groups responded to PE induced vessel contraction equally (FIG. 14A), significant differences in endothelial dependent vessel relaxation occurred between groups, with relaxation segregating with mtDNA haplotype (FIG. 14B). No significant differences were observed in endothelial independent vessel relaxation (FIG. 14C). These data support the notion that the mtDNA haplotype can contribute significantly to endothelial dependent vessel function, and moreover, are consistent with the hypothesis that mitochondrial function and genetics are important factors in influencing individual CVD susceptibility.

Based upon their known mtDNA sequences, phylogenetic analyses were performed to determine the potential phylogenetic relationships among strains of mice that were known to have different susceptibilities to CVD development. FIG. 15 shows a mtDNA phylogeny with indication of relative CVD susceptibilities (susceptible, resistant, very resistant) for each strain of mouse. The mtDNA phylogenetic relationships appear to reflect the relative susceptibility to CVD development, consistent with the hypothesis that mitochondrial genetics plays a role in CVD susceptibility.

In summary, these data collectively show that the CVD resistant C3H/HeN mouse has mitochondrial characteristics that make it less susceptible to cardiovascular disease. Further, these studies show that mitochondrial function and genetics are important factors in influencing individual CVD susceptibility.