U.S. patent application number 12/486375 was filed with the patent office on 2009-12-31 for mitochondrial-nuclear exchanged cells, tissues, organs and animals.
This patent application is currently assigned to The UAB Research Foundation. Invention is credited to Scott Webster Ballinger, Larry W. Johnson, Robert Allen Kesterson, Danny R. Welch.
Application Number | 20090328241 12/486375 |
Document ID | / |
Family ID | 41449367 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090328241 |
Kind Code |
A1 |
Ballinger; Scott Webster ;
et al. |
December 31, 2009 |
MITOCHONDRIAL-NUCLEAR EXCHANGED CELLS, TISSUES, ORGANS AND
ANIMALS
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) ; Welch; Danny R.; (Vestavia
Hills, AL) ; Kesterson; Robert Allen; (Birmingham,
AL) ; Johnson; Larry W.; (Jasper, AL) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O BOX 1022
Minneapolis
MN
55440-1022
US
|
Assignee: |
The UAB Research Foundation
Birmingham
AL
|
Family ID: |
41449367 |
Appl. No.: |
12/486375 |
Filed: |
June 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61076260 |
Jun 27, 2008 |
|
|
|
Current U.S.
Class: |
800/3 ; 435/29;
435/325; 800/13; 800/18; 800/24 |
Current CPC
Class: |
A01K 2227/10 20130101;
A01K 67/0273 20130101; A01K 2217/00 20130101; A61K 49/0008
20130101; A01K 2227/105 20130101; A01K 2267/0375 20130101; A01K
67/0271 20130101; A61D 19/04 20130101; A01K 67/0275 20130101; G01N
33/5023 20130101; A01K 2267/0331 20130101 |
Class at
Publication: |
800/3 ; 435/325;
800/13; 800/18; 800/24; 435/29 |
International
Class: |
G01N 33/00 20060101
G01N033/00; C12N 5/00 20060101 C12N005/00; A01K 67/00 20060101
A01K067/00; C12N 15/00 20060101 C12N015/00; C12Q 1/02 20060101
C12Q001/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] 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.
Claims
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
[0001] 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.
BACKGROUND
[0003] 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
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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
[0008] 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.
[0009] FIG. 2A shows a female mitochondrial-nuclear exchanged mouse
[C57 mtDNA/FVB/N-TgN(MMTVPyMT) nuclear DNA (nDNA)] at 3 months of
age.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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).
[0014] 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.
[0015] FIG. 5 is a schematic summarizing the process of creating
mitochondrial-nuclear exchanged animals.
[0016] 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).
[0017] 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.
[0018] 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.
[0019] 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.degree. 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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 .mu.g 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).
[0024] 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.
[0025] FIGS. 14A, 14B and 14C are graphs showing isometric tension
measured in aortic segments from 14-week old male
C57.sup.mtDNA/C57.sup.nDNA; C.sub.3H.sup.mtDNA/C3H.sup.nDNA;
C3H.sup.mDNA/C57.sup.nDNA; and C57.sup.mtDNA/C3H.sup.nDNA 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
C57.sup.mtDNA/C57.sup.nDNA and C57.sup.mtDNA/C3H.sup.nDNA,
respectively.
[0026] 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
[0027] 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.
[0028] 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).
[0029] 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).
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] FVB/N-TgN(MMTV-PyMT) mice carrying the (MMTV-PyVT) transgene
are susceptible to cancer.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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 .mu.m2
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.
[0053] 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.
[0054] 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.
[0055] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application.
[0056] 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
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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-US-00001 TABLE 1 A summary of the study from the first four
mtDNAC57BL/6::nDNAPyMT mice produced. Onset of Primary Number of
Lung Gender Age Breast Tumor Metastases MNX Female.sup.a 110 days
at sacrifice 79 days 3 MNX Male.sup.a 238 days at sacrifice 107
days None detected MNX Male.sup.a Currenlty 308 days, not None
detected ND yet sacrificed MNX Male.sup.a Currently 308 days, not
None detected ND yet sacrificed Female controls.sup.b 94 .+-. 2.35
days at 67.22 .+-. 3.07 days 24.63 .+-. 5.39 sacrifice Male
controls.sup.b,c Average age at 83 .+-. 20 days Specific number not
sacrifice not provided reported, multiple, 80% penetrance ND--not
determined .sup.amtDNA.sup.C57B1/6::nDNA.sup.PyMT mice
.sup.bmtDNA.sup.FVB/N::nDNA.sup.PyMT mice; Control data are listed
as mean + SEM .sup.cMale control data are from Hazen and Heinecke,
J. Clin. Invest. 99: 2075-2081 (1997).
[0063] "Wild-type" (mtDNA.sup.FVB/N::nDNA.sup.PyMT) mice develop
mammary tumors and metastases with 100% penetrance within 70 days
and 90 days, respectively. The MNX female mouse
(mtDNAC.sup.57BL/6::nDNA.sup.PyMT) 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
(mtDNAC.sup.57BL/6::nDNA.sup.PyMT) 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.
[0064] 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
[0065] 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
[0066] 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.
[0067] 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
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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
.mu.m) 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.
[0074] 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.
[0075] 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 H.sub.2O.sub.2). FIG. 12 shows
that C57 mitochondria exhibited significantly higher levels of
fluorescence compared to C3H mitochondria, consistent with
increased oxidant production.
[0076] 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.
[0077] Because data suggested that C57 mitochondria were more
tightly coupled than C3H, and that higher oxidant production
(presumably O.sub.2-) 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.
[0078] 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 C3H.sup.mtDNA/C57.sup.nDNA and
C57.sup.mtDNA/C3H.sup.nDNA mice were used in vessel relaxation
studies and compared to age-matched wild-type C57BL/6
(C57.sup.mtDNA/C57.sup.nDNA) and C3H/HeN
(C3H.sup.mtDNA/C3H.sup.nDNA) 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.
[0079] 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.
[0080] 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.
* * * * *
References