U.S. patent application number 14/506674 was filed with the patent office on 2015-03-12 for transgenic mouse expressing human lipoprotein (a) with disabled vitamin c gene and its use as a disease treatment model.
This patent application is currently assigned to MATTHIAS W. RATH. The applicant listed for this patent is JOHN CHANG-EUN CHA, Aleksandra Niedzwiecki, MATTHIAS W. RATH. Invention is credited to JOHN CHANG-EUN CHA, Aleksandra Niedzwiecki, MATTHIAS W. RATH.
Application Number | 20150074835 14/506674 |
Document ID | / |
Family ID | 52626914 |
Filed Date | 2015-03-12 |
United States Patent
Application |
20150074835 |
Kind Code |
A1 |
RATH; MATTHIAS W. ; et
al. |
March 12, 2015 |
TRANSGENIC MOUSE EXPRESSING HUMAN LIPOPROTEIN (A) WITH DISABLED
VITAMIN C GENE AND ITS USE AS A DISEASE TREATMENT MODEL
Abstract
The invention discloses novel model of transgenic mammal, a
method of crossbreeding transgenic mammal and the use of the
transgenic mammal for assessing prevention and/or treatment methods
for cardiovascular and other diseases related to lipoprotein(a).
The transgenic mammal expresses human apolipoprotein (a) (apo(a))
and human apolipoprotein B-100 (apo B-100) genes and produces human
lipoprotein (a), apo (a) and apo B-100 and produces no vitamin C.
This novel dual transgenic mammal is the ideal model for testing
pharmaceutical compounds for efficacy and usefulness in the
prevention and/or treatment of human diseases.
Inventors: |
RATH; MATTHIAS W.; (APTOS,
CA) ; Niedzwiecki; Aleksandra; (APTOS, CA) ;
CHA; JOHN CHANG-EUN; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RATH; MATTHIAS W.
Niedzwiecki; Aleksandra
CHA; JOHN CHANG-EUN |
APTOS
APTOS
San Mateo |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
RATH; MATTHIAS W.
Aptos
CA
|
Family ID: |
52626914 |
Appl. No.: |
14/506674 |
Filed: |
October 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14025532 |
Sep 12, 2013 |
|
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14506674 |
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Current U.S.
Class: |
800/3 ; 800/22;
800/9 |
Current CPC
Class: |
A01K 2217/15 20130101;
A01K 2267/0368 20130101; C12N 15/8509 20130101; A01K 2207/15
20130101; A01K 2267/03 20130101; A01K 2217/052 20130101; C12N
9/0006 20130101; A01K 67/0278 20130101; A01K 2267/0375 20130101;
C12N 15/90 20130101; A01K 2227/105 20130101; A01K 2267/0337
20130101; A01K 67/0276 20130101; A01K 67/0275 20130101; C12Y
101/03008 20130101; A01K 2217/00 20130101; A01K 2217/05 20130101;
A01K 2267/0362 20130101; A61K 49/0008 20130101; C07K 14/775
20130101; G01N 33/92 20130101 |
Class at
Publication: |
800/3 ; 800/9;
800/22 |
International
Class: |
A01K 67/027 20060101
A01K067/027; A61K 49/00 20060101 A61K049/00 |
Claims
1. A dual transgenic mammal, comprising: a third strain of
transgenic mammal was obtained by crossbreeding a first knockout
strain mammal and a second strain of transgenic mammal expressing a
set of genes that are human in nature, wherein the first knockout
strain mammal is lacking a gulonolactone oxidase gene, GULO gene
(GULO-/-), and produces no Vitamin C, wherein the second strain of
mammal possesses a human apolipoprotein (a) gene (apo (a)+),
wherein the third strain of transgenic mammal expresses the human
apo (a) gene (apo (a)+) and, simultaneously lacks the GULO gene
(GULO-/-); and a fifth strain of mammal made by crossbreeding the
first knockout strain mammal lacking the GULO gene and the fourth
strain of mammal possessing a human apolipoprotein B-100 gene (apo
B-100+), wherein the fifth strain of mammal lacks the GULO gene
(GULO-/-) and expresses the human apo B-100 gene (apo B-100+).
2. The transgenic mammal of claim 1, further comprising: a dual
transgenic mammal that lacks the GULO gene (GULO-/-) and,
simultaneously, produces human lipoprotein (a) (Lp(a)+) was
obtained by crossbreeding the third strain of mammal lacking GULO
gene (GULO-/-) and, simultaneously expressing the human apo (a)
gene (apo (a)+), and the fifth strain of transgenic mammal lacking
the GULO gene (GULO-/-) and, simultaneously, possessing the human
apo B-100 gene (apo (B-100)+).
3. The transgenic mammal of claim 3, wherein the first knockout
mammal, second, third, fourth, fifth transgenic mammal and the dual
transgenic mammal is a mouse.
4. A method, comprising: crossbreeding a first knockout strain and
a second strain of mouse to obtain and a third strain of transgenic
mouse that do not produce vitamin C and expresses a first human
gene to produce the human products; crossbreeding the first
knockout strain and a third strain of mouse to produce a fifth
transgenic mouse to express a second human gene and produce no
vitamin C; and crossbreeding the fifth strain of transgenic mouse
and the third strain of transgenic mouse to produce novel dual
transgenic mouse that expresses the first human gene, the second
human gene and a third human gene to make a first product, second
product and a third product but produce no vitamin C.
5. The method of claim 4, further comprising: testing the
transgenic mouse using a specific sequence of primer to confirm the
presence of the two human genes, human apo (a) and human apo B-100,
the first product, second product, third product and lack of GULO
gene.
6. The method of claim 4, wherein the first strain of transgenic
mouse lacks the GULO gene (GULO-/-), the second strain of
transgenic mouse expresses human apo (a) (+), the third strain
lacks the GULO gene (GULO-/-) and expresses human apo (a) (apo
(a)+), the fourth strain of transgenic mouse expresses human apo
B-100 (B-100+), the fifth strain lacks the GULO gene (GULO-/-) and,
simultaneously, expresses human apo B-100 (B-100+).
7. The method of claim 5, wherein the specific sequences are Seq 1
to Seq 12.
8. The method of claim 4, further comprising: crossbreeding the
third strain expressing human GULO (GULO-/-) and human (apo (a)+),
and the fifth strain of transgenic mouse expressing human GULO
(GULO-/-) and human apo B-100(B-100+) to obtain a dual transgenic
mouse.
9. The method of claim 9, wherein the dual transgenic mouse lack
the ability to express the gulonolactone oxidase gene [GULO
(GULO-/-)] and produces human lipoprotein (a) (Lp(a)).
10. The method of claim 6, wherein the first product is human apo
(a), second product is human apo B-100 and the third product is
human Lp(a).
11. The method of claim 9, further comprising: testing an effect of
a pharmaceutical drug that will affect Lp(a) synthesis and/or Lp(a)
plasma levels and/or Lp(a) deposition inside the vascular wall in
the transgenic mouse with the purpose to treat a cardiovascular
disease.
12. A method, comprising: crossbreeding a dual transgenic mouse to
produce human Lp(a) and not produce Vitamin C due to lack of GULO
(GULO-/-) gene using transgenic mouse having a first knockout
strain, a second strain to make a third strain and using the first
knockout strain and fourth strain to make a fifth strain, using the
third strain and the fifth strain to make a dual transgenic mouse;
and treating the dual transgenic mouse with Lp(a)-modulating
compounds in order to identify preventive and/or therapeutic
approaches for a human Lp(a)-related diseases.
13. The method of claim 12, wherein the first knockout strain of
transgenic mouse lacking the GULO gene (GULO-/-), the second strain
of transgenic mouse expresses human apo (a) (apo(a)+), the third
strain lacks the GULO gene (GULO-/-) and, simultaneously, possesses
human apo(a) (apo (a)+) gene, the fourth strain of transgenic mouse
possesses human apo B-100 (B-100+), the fifth strain lacks the GULO
gene (GULO-/-) and, simultaneously, expresses human apo B-100
(B-100+).
14. The method of claim 13, wherein the first knockout strain does
not produce Vitamin C.
15. The method of claim 13, wherein the second strain of transgenic
mouse produces human apo (a).
16. The method of claim 13, wherein the third strain of transgenic
mice does not produce Vitamin C and produces human apo (a).
17. The method of claim 13, wherein the fifth strain of transgenic
mouse does not produce Vitamin C and produces human apo B-100.
18. The method of claim 12, wherein the dual transgenic mouse lacks
the GULO gene and does not produce Vitamin C and, simultaneously
produces human Lp(a).
19. The method of claim 12, wherein the human Lp(a)-related disease
is a cardiovascular disease.
20. The method of claim 12, wherein the human Lp(a)-related disease
is inflammatory, infectious or degenerative in nature.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This instant application is a continuation of U.S. Utility
application Ser. No. 14/025,532 filed on Sep. 12, 2013. The pending
and now allowed U.S. Utility application Ser. No. 14/025,532 is
hereby incorporated by reference in its entireties for all of its
teachings.
FIELD OF TECHNOLOGY
[0002] This disclosure relates generally to a transgenic mouse that
has been genetically altered to express human lipoprotein (a) and a
disabled gene for the expression of Vitamin C. More specifically
the two defining human proteins of lipoprotein (a), apolipoprotein
(a) and apolipoprotein B-100 gene may be expressed either
individually or in combination without the expression of Vitamin C
gene. This application contains sequence listing that has been
submitted as an ASCII file named RIPLLC018017US1sequence_ST25, the
date of creation Sep. 12, 2013, and the size of the ASCII text file
in bytes is 2 kb. The dual transgenic mouse embryo referred to as
Rath M Human Lipoprotein(a);Gulo(-/-) has the Jackson Stock#
912329, having been deposited in The Jackson Laboratory on Apr. 8,
2013.
BACKGROUND OF THE INVENTION
[0003] Cardiovascular disease is responsible for half of the deaths
in the industrial world. Over the past decades a new risk factor
for this disease has emerged, lipoprotein (a)(Lp (a)). Lp (a) is
has been shown to be an independent risk factor for myocardial
infarctions, (Rhoads G G et. al., 1986, Clarke et al, 2009)
cerebrovascular disease (Zenker G et. al.,1986) and other forms of
cardiovascular disease. Furthermore, Lp(a) has been identified as a
significant component of human atherosclerotic plaques (Rath M et.
al., 1989). Aside from Niacin, there is currently no accepted
effective treatment available in clinical cardiology to lower Lp(a)
plasma levels or to prevent its deposition inside the vascular
wall.
[0004] Lp(a) was discovered by Kare Berg in 1963 (Berg, K et. al.,
1963). It is composed of a low-density-lipoprotein molecule (LDL)
and apolipoprotein (a) (apo(a)), a glycoprotein attached to the
structural protein of LDL, apolipoprotein B-100 (apo B), via
disulfide bonds. The cDNA of apo(a) shows a strong homology with
plasminogen containing multiple repeats of plasminogen kringle IV.
Due to this homology apo(a) binds to fibrinogen/fibrin and
attenuates fibrinolysis (McLean, J W et. al. 1987).
[0005] Lp(a) is primarily found in humans and subhuman primates and
the appearance of the apo(a) gene was dated to about 40 million
years ago, about the time of the divergence of the Old World and
New World monkeys (McLean, et al. Nature, 1987). This was also the
time point during evolution when the ancestor of man lost the
ability for endogenous ascorbate synthesis due to a mutation in the
gene encoding for gulonolactone oxidase (GULO), an essential enzyme
for the conversion of glucose to ascorbate (vitamin C) (Chatterjee
I B, 1973, Nikishimi M et al., 1991).
[0006] The significance of ascorbate deficiency in initiating the
process of atherogenesis has recently been documented in mouse
unable to express the gene for L-gulonolactone oxidase (GULO-/-)
(Maeda N et. al., 2000).
[0007] Roy et. al. (2003, U.S. Pat. No. 6,512,161) discusses
several failed attempts to create animal models for expressing
specifically Lp(a) in models such as rats, mouse and guinea pigs
and state that they don't always represent human metabolism and
human-related diseases. In their study they invented a rabbit model
expressing human apo (a) and human apo B-100 genes. However, the
transgenic rabbit developed by Roy et. al. (2003) also does not
mimic the human physiology with respect to another key metabolic
aspect: unlike humans, rabbits are able to produce their own
Vitamin C.
[0008] There exists a need for a dual transgenic mammal model
displaying these unique genetic features in order to develop new
preventive and therapeutic approaches related to them.
SUMMARY
[0009] The current application discloses a method of making and
using a dual transgenic mammal (mouse, rat and other mammalian
species) that possesses the genes for human apo (a) and/or apo
B-100 and produces human (Lp(a) while, at the same time, being
unable to produce vitamin C. In one embodiment, a third strain of
transgenic mammal was obtained by crossbreeding the first knockout
strain mammal and second strain of transgenic mammal (may be a
mouse or other animals) expressing a set of genes that are human in
nature, wherein the first strain is a knockout mammal possessing a
non-functional L-gulonolactone oxidase (GULO) (GULO-/-) and hence
produces no Vitamin C, wherein the second strain of mice expresses
human apo (a) gene (apo (a)+) and produces apolipoprotein (a)(apo
(a)), wherein the third strain of transgenic mice possesses
non-functional L-gulonolactone oxidase (GULO-/-) gene and a
functional human apo (a) gene (apo (a)+). Hence this third strain
of mammal will not produce vitamin C but will produce human apo
(a).
[0010] In another embodiment, a fifth strain of transgenic mammal
was made by crossbreeding the first knockout strain mammal
possessing non-functional L-gulonolactone oxidase (GULO-/-) gene
and the fourth strain expressing human apo B-100 gene (apo
(B-100)+), wherein the fifth strain of mammal possesses
non-functional L-gulonolactone oxidase (GULO-/-) gene and a
functional human apo B-100 gene (apo (B-100)+). Hence the fifth
strain of transgenic mammal will not produce vitamin C and will
produce human apolipoprotein B-100.
[0011] In another embodiment, third strain and fifth strain of
transgenic mammal were crossbred to obtain a novel dual transgenic
mammal (may be a mouse for example) that had a knockout GULO gene
(GULO-/-), a functional human apo B-100 gene (apo(B-100)+) and a
functional human apo (a) gene (apo (a)+). The novel dual transgenic
mammal will hence, produce apolipoprotein (a)(apo (a)) and/or
apolipoprotein B-100 (apo B-100) as well as the complete
lipoprotein(a) particle (Lp(a) and will not produce vitamin C. This
novel double transgenic mammal model resembles the human system
with respect to the inability of endogenous ascorbate synthesis
and, congruently, the expression of apo(a), apo (B-100) as well as
the complete lipoprotein(a) particle (Lp(a)). In the instant
disclosure a mouse model is used but other mammals may be used and
crossbreeding, insertion of genes or deletion of genes may be done
to produce these dual transgenic mammals to express or suppress
human genes in any combination.
[0012] In one embodiment, a mammal, a mouse whose genome lacks the
ability for endogenous ascorbate synthesis
and--simultaneously--expresses a human apo (a) is disclosed. In
another embodiment, a dual transgenic mammal, a mouse, whose genome
lacks the ability for endogenous ascorbate synthesis
and--simultaneously--expresses a human apo B-100 is disclosed. In
another embodiment, a transgenic mammal, a mouse, whose genome
lacks the ability for endogenous ascorbate synthesis
and--simultaneously--produces a human Lp(a) is disclosed. An animal
model may be created by crossbreeding, gene insertion or other
methods of molecular biology and/or genetic engineering.
[0013] In one embodiment, the novel dual transgenic mouse to be
used as a model for cardio vascular disease (CVD) study. In another
embodiment, the dual transgenic mice to be used as a model for
treating CVD like diseases. In another embodiment, the dual
transgenic mouse may be used as a model to test new and old drugs
to treat diseases associated with Lp(a) synthesis and lack of
vitamin C production.
[0014] In one embodiment, the dual transgenic mouse model may be
used for testing effect of various drugs involved in ischemic heart
disease, cardiovascular diseases, including coronary artery
disease, cerebrovascular disease (stroke), renal vascular disease,
peripheral vascular disease, aneurysms, thrombotic conditions,
other forms of vascular disease, inflammatory conditions, as well
as infectious diseases, neuroinflammatory and neurodegenerative
diseases.
[0015] In one embodiment, a process for making a dual transgenic
mammal which lacks the ability for endogenous ascorbate synthesis
and--simultaneously--is capable of producing human
apolipoprotein(a) or human apolipoprotein B or Lp(a) comprising
mating a first mammal in which the ability for ascorbate synthesis
has been genetically deleted with a second mammal which has a
genome encoding human apo (a) or human apo B-100 or both of these
apolipoproteins in such a manner that they combine in vivo in said
transgenic mammal to produce the complete human lipoprotein(a)
particle (Lp(a)).
[0016] In another embodiment, a method for determining whether a
compound can treat atherosclerosis or an undesirable plasma lipid
profile comprising: a) comparing the lipid profile or state of
atherosclerosis in a first transgenic mammal fed a diet generally
known to be atherogenic and treated with said compound, to the
lipid profile or state of atherosclerosis in a second transgenic
mammal fed the same atherogenic diet but not treated with said
compound; and determining the potential therapeutic effect of said
compound based upon comparative evaluation of the lipid profile or
state of atherosclerosis in said first and second transgenic
mammal; wherein said first and second transgenic mammal each being
a transgenic mammal.
[0017] In one embodiment, a treatment method wherein the drug to
treat the effect of high Lp(a) in the absence or in presence of
micronutrients such as vitamin C is observed using transgenic mouse
that does not produce vitamin C and produces human Lp(a).
[0018] The composition, method, and treatment disclosed herein may
be implemented in any means for achieving various aspects, and may
be executed in a form suitable for the mammal.
BRIEF DESCRIPTION OF DRAWINGS
[0019] Example embodiments are illustrated by way of example and
not limitation in the Figures of the accompanying drawings, in
which like references indicate similar elements and in which:
[0020] FIG. 1 shows the scheme used for creating the dual
transgenic mouse.
[0021] FIG. 2 shows the expression of disulfide linked human apo(a)
and human apoB-100 as assembled Lp(a) with a lipid globule in male
and female transgenic mouse with both human apo(a) and human apo
B-100 genes, but not in male and female transgenic mouse without
both genes.
[0022] FIG. 3 shows the expression of human apo(a) protein in
female and male mice.
[0023] FIG. 4 shows the expression of human apo B-100 protein in
female and male transgenic mouse in LDL particles and Lp(a)
particles.
[0024] FIG. 5 shows the serum ascorbate level in transgenic female
and male mouse (26-29 weeks old) supplemented with Vitamin C or
deprived of Vitamin C in micromoles/liter (uM).
[0025] FIG. 6 shows Serum Protein Immunofixation Electrophoresis
(SPIFE) cholesterol profile of hypoascorbemic or fully
ascorbate-supplemented Lp(a)+GULO (-/-) mouse.
[0026] FIG. 7 shows Immunofixation Electrophoresis (IFE)
apo(a)-particle profile of hypoascorbemic or fully
ascorbate-supplemented Lp(a)+GULO (-/-) mouse.
[0027] FIG. 8 shows IFE Human Apo B-Particle profile of
hypoascorbemic or fully ascorbate-supplemented Lp(a)+GULO (-/-)
mouse.
[0028] Other features of the present embodiments will be apparent
from the accompanying drawings and from the detailed description
that follows.
DETAILED DESCRIPTION
[0029] The invention discloses novel dual transgenic mammal/mouse,
method of crossbreeding a dual transgenic mammal (may be a mouse or
other animals) and the use of the dual transgenic mammal/mouse for
assessing treatment method for cardiovascular and related diseases.
The dual transgenic mouse expresses human apolipoprotein (a) and
apolipoprotein B-100 genes and produces apolipoprotein (a) and
apolipoprotein B-100 as well as complete human lipoprotein (a)
particles in this mouse which, simultaneously, does not express
L-gulonolactone oxidase (GULO -/-) and, consequently, does not
produce vitamin C. This novel dual transgenic mouse is the ideal
model for testing pharmaceutical compounds for treatment efficacy
and usefulness for Lp(a) modulation with a variety of biological
and/or pharmaceutical compounds, including but not limited to,
nutrition, pharmaceutical drugs and treatment methods that affect
human beings.
[0030] Although the present embodiments have been described with
reference to specific example embodiments, it will be evident that
various modifications and changes may be made to these embodiments
without departing from the broader spirit and scope of the various
embodiments.
Cross Breeding of Mammal/Mouse:
[0031] An animal model may be created by crossbreeding, gene
insertion or other methods of molecular biology and/or genetic
engineering. In an exemplary example crossbreeding of a knock mouse
and a specific human expressing gene containing mouse is disclosed
to create a dual transgenic mouse.
[0032] BALB/cBy-Gulo (-/-) mouse: The strain,
BALB/cBy-Gulo.sup.sfx/J was a spontaneous mutation, mapped to the
Gulonolactone oxidase locus, the gene for vitamin C synthesis. The
GULO (-/-) strain mouse (the first knockout strain of transgenic
mouse) was generated from heterozygous (hemizygous) GULO (+/-)
breeders obtained from The Jackson Laboratory (Table 1). The mouse
was bred under vitamin C supplementation until an adequate number
of homozygous GULO (-/-) breeders were obtained.
TABLE-US-00001 TABLE 1 GULO (-/-) strain description: Allele Symbol
Gulo.sup.sfx Allele Name spontaneous fracture Allele Type
Spontaneous Strain of Origin BALB/cBy-Rasa3 Gene Symbol and Name
Gulo, gulonolactone (L-) oxidase Chromosome 14 Gene Common Name(s)
AU018375; BC028822; L-gulono- gamma-lactone oxidase; MGC: 29968;
MGC: 37793; MGC: 37880; cDNA sequence BC028822; expressed sequence
AU018375; sfx; spontaneous fracture; General Note This spontaneous
mutation appeared in a BALB/cBy-scat colony at The Jackson
Laboratory. The scat and sfx mutations were separated from each
other by backcrossing BALB/cBy-scat mouse to BALB/cBy mouse and
observing F2 offspring for those that exhibited the sfx phenotype
but not the scat phenotype. Molecular Note The mutation in the sfx
mouse is a deletion that includes the entire Gulo gene. [MGI Ref ID
J: 95128]
[0033] Maintenance of GULO (-/-) mouse: GULO (-/-) mouse are unable
to synthesize their own vitamin C; therefore this nutrient needs to
be present in the mouse diet. Vitamin C was provided in a double
distilled drinking water containing 150 mg/L ascorbic acid (Sigma)
and 0.01 mM EDTA (Sigma) to stabilize vitamin C from degradation by
interaction with trace metals. The water also contained 10 g/L of
sucrose in order to mask a taste offensive to mouse. Water was
changed twice a week. In addition, these mouse received food
fortified with 500 ppm L ascorbyl-polyphosphate, the standard
veterinary feed source of stable vitamin C milled at Test Diet as
Modified Custom Lab Diet #5A38.
[0034] Human Apo (a) Mouse:
[0035] The Human apo(a) mouse (second strain of transgenic mouse)
was obtained from the Mutant Mouse Regional Resource Centers
(MMRRC), supported by the NIH. The strain, FVB/N-Tg(LPA, LPAL2,
PLG)1Hgc/Mmmh, was created using a 270 kb YAC that harbors human
apo(a) and apo(a) like and plasminogen genes. The donor was Edward
M. Rubin, M.D., Ph.D., Lawrence Berkeley National Laboratory.
Founder mice were bred until sufficient number of apo(a)+Gulo
wildtype mice were obtained for crossbreeding. Genotyping for the
transmission and presence of the transgene was performed at
Transnetyx (Cordova, TN) upon tail clip tissue and transgenic
mutants confirmed positive for apo(a) in the genome selected for
cross-breeding.
[0036] Human Apo B-100 Mouse:
[0037] The human apoB-100 mouse (fourth strain of transgenic
mammal/mouse) was obtained from Taconic Farms, Inc. under academic
research agreement. The strain, B6.SJL-Tg(APOB)1102Sgy N20+?, or
apoB-100 mouse, was developed by MacRae F. Linton et. al. of the
Gladstone Institute of Cardiovascular Disease by microinjecting the
human apolipoprotein B100 gene into C57BL/6J.times.SJL zygotes. The
resultant mouse was backcrossed to C57BL/6 for 4 generations (N4).
Taconic received stock from Xenogen Biosciences in May 1996. The
mouse was maintained by backcrossing hemizygous Apo (B-100) mouse
with C57BL/6NTac inbred mouse. Hemizygous mouse were bred to obtain
homozygous Apo (B-100) mouse. Genotyping for transmission and
presence of the transgene Human Apo (B-100) in the genome was
performed at Transnetyx upon tail clip tissue and transgenic
mutants selected for cross-breeding.
[0038] Cross Breeding Steps Leading to Generating a Mouse Strain
Producing Human Lp(a):
[0039] FIG. 1 shows the various steps used for crossbreeding
several strains of mouse to obtain a dual transgenic mouse. The
terms mouse and mice are used interchangeably and they all mean
mouse in the instant specification. Crossbreeding (108) of human
apo (a) mice (104) (the second strain of mammal)+GULO (-/-) (102)
(a first knockout strain mammal) mice/mouse to produce a third
strain of transgenic mammal/mouse that expresses the human
apolipoprotein (a) gene (apo (a)+) and, simultaneously lacks the
GULO gene (GULO-/-) (112) was performed. A GULO (-/-) (102) (a
first knockout strain mammal) and human apo B-100 mouse represented
as fourth strain of mammal (106) was crossbred (108) to produce a
fifth strain of mouse that the fifth strain of mammal lacks the
GULO gene (GULO-/-) and expresses the human apolipoprotein B-100
gene (apo (B-100)+) (114). Two transgenic founding strains were
thus obtained for further crossbreeding: [0040] Human apo(a)+GULO
(-/-) mouse--third strain of transgenic mouse [0041] Human apo
B-100+GULO (-/-)mouse--fifth strain of transgenic mouse.
[0042] Crossbreeding (108) the Founding Strains for Obtaining Mouse
Strain:Human Lp(a)+GULO (-/-) (116):
[0043] Newly generated mouse breeders of human apo(a)+GULO (-/-)
mouse (112) and human apo B100+GULO (-/-) (114) were subsequently
crossed (108) with one another to generate the new mouse strain:
human Lp(a)+GULO (-/-) mice (116) which had human apo(a)+human
ApoB-100+GULO (-/-), named as "Rath M Human Lipoprotein(a);Gulo
(-/-)" strain (116). The dual transgenic mouse embryo referred to
as Rath M Human Lipoprotein(a);Gulo(-/-) has the Jackson Stock#
912329, having been deposited in The Jackson Laboratory on Apr. 8,
2013.
Genotyping:
[0044] Genotyping for the GULO locus and its homozygosity was
performed via Taqman FAM Probe Real Time-PCR at Transnetyx upon
tail clip tissue derived DNA obtained using standard DNA isolation
and PCR techniques. Transgene presence for human apo B-100 and
human apo(a) were also conducted at Transnetyx.
[0045] Tail clips were obtained from mouse under anesthesia and
then shipped to Transnetyx where the following probe sets were
designed and used for Real-Time PCR detection of genomic DNA
presence or absence (primers used are shown below in the Tables 2,
3 and 4. Litters were genotyped via tail clip Taqman PCR at
Transnetyx and those positive for genomic transgenes apo(a) and
apoB-100, as well as homozygous knockout mutation for the
L-gulonolactone-oxidase gene, GULO (-/-), which indicates vitamin C
synthesis defect, were selected and labeled as "Rath M Human
Lipoprotein(a); Gulo (-/-)" founder mouse. In FIG. 2 we show that
the disulfide linked lipoprotein (a) is formed in the dual
transgenic mouse and shown as Lp(a) GULOKO F1, F2, M1 and M2.
Similarly, the lack of Lp(a) in apo(a)+mouse without human apoB-100
expression and apo B-100 +mice without apo(a) expression is also
shown.
TABLE-US-00002 TABLE 2 GULO testing: Gulo-1 KO
CTAGTGTAGTCTAGGTGATAAGGATCAACT--Seq 1 Forward Primer: Gulo-1 KO
CAGCTCAGAGAGAGAATGAATCACA--Seq 2 Reverse Primer: Reporter 1:
CTGACATCCCTTAGGAGTTC--Seq 3 Gulo-1 WT AGATGTGTTCCAGGCTGCAA--Seq 4
Forward Primer: Gulo-1 WT CACACACTGCAGGGTGACA--Seq 5 Reverse
Primer: Reporter 1: CTGCCTGGGTGTTATC--Seq 6
[0046] Genotype Results Interpretation: Gulo-1 KO+, Gulo-1
WT+=Hemizygous Vitamin C generating mouse. Gulo-1 KO-, Gulo-1
WT+=Homozygous wild type Vitamin C generating mouse. Gulo-1 KO+,
Gulo-1 WT-=Homozygous Vitamin C defective mouse.
[0047] Mouse homozygous for the knockout, GULO(-/-) mouse was
selected for cross-breeding.
TABLE-US-00003 TABLE 3 B: Human apo(a) testing HuLPA-1 Tg (Human
CACTACATTTTGTGCCAGAGATGGA--Seq 7 apo(a) transgene) Forward Primer:
HuLPA-1 Tg CCCTGTCCTGAGGCTCCTTA--Seq 8 Reverse Primer: Reporter 1:
TCAGCAGCCCTCTTCC--Seq 9
[0048] Genotype Results Interpretation: +=Human apo(a) gene
positive. -=Human apo(a) gene negative. Mouse positive for the
transgene were selected for cross-breeding.
TABLE-US-00004 TABLE 4 Human apo B-100 testing primers ApoB Tg
(Human AGGTTTAACTCCTCCTACCTCCAA--Seq 10 ApoB100 Transgene) Forward
Primer: ApoB Tg TGAGGGAGAGGGTTCCATCTT--Seq 11 Reverse Primer:
Reporter 1: ACCAGATAACAGGAAGATATG--Seq 12
[0049] Genotype Results Interpretation: +=Human apoB-100 gene
positive. -=Human apoB-100 gene negative. Mouse positive for the
transgene were selected for cross-breeding.
[0050] The genotype of the Lp(a); GULO (-/-) mouse is denoted as h
apo(a)+; h apoB-100+; GULO(-/-). The mouse must continually be
maintained on vitamin C supplementation as described above.
[0051] Confirming the transgene mouse generation at the level of
protein: The presence of human apo(a) and human apo B-100 proteins
in mouse sera was determined by ELISA in the serum drawn from the
GULO (-/-) mouse, the apo(a)+GULO(-/-) mouse, the apoB+GULO (-/-)
mouse, and the Lp(a)+GULO (-/-) mouse.
TABLE-US-00005 TABLE 5 Lp(a); GULOKO mice and hApoB; GULOKO mice
express human ApoB via AssayPro ApoB ELISA Sample name ug/mL ug/ml
* 20000 mg/dL gko m1 0 gko m2 0.001153 gko f1 0.000374 gko f2
-0.00036 Lp(a); gko m1 0.014717 294.3472224 29.4 mg/dL Lp(a); gko
m2 0.015867 317.346142 31.7 mg/dL Lp(a); gko f1 0.041693
833.8570197 83.3 mg/dL Lp(a); gko f2 0.041411 828.2183041 82.8
mg/dL apo(a); gko m1 0.000185 apo(a); gko m1 -0.00018 apo(a); gko
f1 -0.00054 apo(a); gko f2 -0.00018 hApoB; gko m1 0.056842
1136.847637 114 mg/dL hApoB; gko m2 0.020853 417.0655494 41.7 mg/dL
hApoB; gko f1 0.003275 65.5092541 6.6 mg/dL hApoB; gko f2 0.017319
346.3860878 34.6 mg/dL Serum diluted 1:20,000
[0052] The apo(a) protein was present in serum of both male and
female mouse before puberty. Male mouse after puberty have
significantly or completely repressed apo(a) protein expression due
to elevated testosterone levels. Apo(a) expression in male mouse
may be restored via castration, continuous infusion of growth
hormone via osmotic pump, or by biochemical modulation by dietary,
chemical, or biological inducers.
[0053] Human apo B-100 protein expression: The presence of human
apoB-100 protein in serum was determined by ELISA in a serum drawn
from the GULO (-/-) mouse, the apo(a); GULO(-/-) mouse, the apoB;
GULO (-/-) mouse, and the Lp(a); GULO (-/-) mouse.
[0054] Presence of apoB-100 protein in mouse serum was determined
by using Assaypro (St. Charles, MO) AssayMax Human Apolipoprotein
enzyme immunoassay which is human apoB-100 specific and does not
cross-react with mouse apoB-100, and/or with any other of the
apolipoproteins (Apo AI, ApoC, ApoE).
[0055] Human apoB-100 was detected in the sera of hApoB-100; GULO
(-/-) mouse, hApoB-100; apo(a); GULO(-/-) mouse, but not apo(a);
GULO (-/-) mouse or GULO (-/-) mouse (Table 5).
[0056] Serum apo(a) protein was present in apo(a) gene containing
GULO (-/-) mouse, apo(a) and human ApoB-100 gene containing GULO
(-/-) mouse, but not GULO (-/-) mouse without the transgene nor in
GULO (-/-) mouse with only human apo B-100 (Table 6)). These
results confirm expression and translation of the human transgene
apo(a) to serum protein apo(a).
TABLE-US-00006 TABLE 6 Female and male apo(a) expression. Male
mouse may have low expression because of high testosterone levels
in blood. Sample names mg/dL apo(a) Comments GKO 0.66168 (below
This test does not cross react with plasminogen or LDL. m1
detection limit) GKO 0 (below detection limit) m2 GKO f1 -0.33084
(below No apo(a) detected in background GULOKO mice. detection
limit) GKO f2 -0.33084 (below No apo(a) detected in hApoB; GULOKO
mice. detection limit) Lp(a); 0.16542 (below Extremely high apo(a)
detected in apo(a); GULOKO gko m1 detection female mice. limit)
Lp(a); 0.16542 (below No apo(a) detected in apo(a); GULOKO male
mice. gko m2 detection limit) Lp(a); 86.51466 (#464) Extremely high
apo(a) detected in Lp(a); GULOKO gko f1 female mice, either
generation F1 or F2. Lp(a); 79.4016 (#110) No apo(a) detected in
Lp(a); GULOKO male mice, either gko f2 F1 or F2. apo(a); 0.33084
(below detection limit) gko m1 apo(a); -0.33084 (below Sex steroid
testosterone suppresses apo(a) production in gko m2 detection these
mice. limit) apo(a); 133.8248 Orchidectomy may have to be performed
in order to gko f1 express Lp(a). apo(a); 156.1565 gko f2 hApoB;
0.16542 (below (#110 - ApoB gene signal = 13.5, apo(a) gene signal
= gko m1 detection 3.7) limit) hApoB; 0 (below (#464 - ApoB gene
signal = 20.4, apo(a) gene signal = gko m2 detection 8.5) limit)
hApoB; -0.16542 (below (#456 - ApoB gene signal = 18.8, apo(a) gene
signal = gko f1 detection 6.4) limit) hApob; 0.66168 (below
detection limit) gko f2
[0057] Human apo (a) protein expression: Presence of apo (a)
protein in serum was determined by using the IBL International GmbH
Lp(a) Enzyme immunoassay which is human apo (a) specific and does
not cross-react with plasminogen or LDL. All known isoforms of
apo(a) can be detected.
[0058] Human Lp(a) protein expression: The Lp(a) particles are
composed of human apo(a) protein linked to human apo B-100 (the
main protein of the LDL particle) by a disulfide bond.
[0059] SPIFE Cholesterol Profiling (FIG. 2): The presence of
complete Lp(a) lipoprotein particles in the Lp(a)+GULO(-/-)
transgenic mouse serum was confirmed using the electrophoresis
method with Helena (Beaumont, Tex.) SPIFE Cholesterol Profiling and
Immunofixation electrophoresis (IFE).
[0060] The Lp(a)-cholesterol band runs at a specific migration
distance in respect to LDL-cholesterol, and HDL-cholesterol, and it
is found in human Lp(a)+; GULO(-/-) mouse sera, but not in the sera
of GULO (-/-), human apo(a); GULO(-/-), or human apoB; GULO (-/-)
mouse confirming that the presence of both human apoB-100 and human
apo(a) are necessary to form complete Lp(a) particles in serum and
that human apo(a) alone is insufficient to produce Lp(a) and it
does not link to mouse LDL via disulfide bonds. In FIG. 2 the band
closest to the top of the gel corresponds to LDL-cholesterol, and
the band furthest from the top to HDL-cholesterol. The tight,
middle bands located between the LDL and HDL bands which are
present in lanes 16-18 represent the Lp(a)-cholesterol from three
different female Lp(a); GULO(-/-) mice. These bands are missing
from those GULO(-/-) mice not simultaneously expressing both human
apo(a) and human apoB-100 transgenes. Lane 19 represents
charge-mass shifts resulting from a 24 hour room temperature
incubation of serum specimen #18, which indicates that a small
shift in the particle migration may relate to lipoprotein
oxidation.
[0061] Immunofixation Electrophoresis (IFE) using the mouse sera
was conducted on individual apo(a) (FIG. 3) containing particles
and human apoB-100 containing particles (FIG. 4) as well, using
human specific apo(a) and apoB-100 antibodies at Health Diagnostic
Laboratory, Inc. (Richmond, Va.). The bands represent apo(a) and
apoB-100 protein respectively in transgenic mouse and visualization
of the same in serum derived from female and male mouse.
[0062] Confirming a lack of vitamin C production in the transgenic
mouse strains: Serum level of ascorbate (vitamin C) in both
GULO(-/-) mouse and a newly generated strain of Lp(a)+GULO (-/-)
depends on its dietary supplementation. Mouse kept on vitamin C
deficient diet has a gradually diminishing serum concentration of
vitamin C until it reaches zero or the mouse dies. Serum levels of
vitamin C were obtained using the Biovision (Mountain View, Calif.)
Ferric Reducing Ascorbate Assay (FRASC) Kit (FIG. 5).
[0063] Lipoprotein-cholesterol, Apo(a) particle, and ApoB-100
particle Modulation (FIG. 6, FIG. 7, and FIG. 8): The analysis was
conducted on mouse sera from the Lpa+Gulo (-/-)mouse supplemented
with either 30 mg/L, 60 mg/L or 150 mg/L of ascorbic acid provided
in drinking water in addition to 500 ppm vitamin C provided in food
(full supplementation). It was observed that the whole spectrum of
lipoprotein cholesterols and/or lipoprotein particles could be
modulated by dietary ascorbate alone. These data give additional
particle number data and in conjunction with the lipoprotein
cholesterol load data provided comprehensive confirmation of the
presence of apo(a) protein, human apoB-100 protein, and disulfide
linked Lp(a) in these mouse sera.
[0064] Sample order in FIGS. 6-8 correspond to the following key,
with 30 vc referring to 30 mg/L Vitamin C group, 60 vc referring to
60 mg/L Vitamin C group, and sc referring to fully supplemented
(150 mg/L Vitamin C+500 ppm Vitamin C in food) control group. The
first three wells of each row were not used.
TABLE-US-00007 TABLE 7 Sample list for the wells in FIG. 6, 7 and
8. Well# Group Sample ID 4 30vc1f e710f 5 30vc2f e689f 6 30vc2f
e598f 7 30vc2f e704f 8 30vc2f e723f 9 30vc2f e717f 10 human control
11 30vc1f fell off 1 12 30vc1f e762f 13 30vc1f e759f 14 30vc1f
e699f 15 30vc1f e706f 16 30vc1f fell off 2 17 60vc2f e773f 24
60vc2f ea315f 25 60vc2f e771f 26 60vc2f e694f 27 60vc2f e761f 28
60vc2f e768f 29 60vc1f e776f 30 human control 31 60vc1f e777f 32
60vc1f e780f 33 60vc1f fell off 34 60vc1f e733f 35 60vc1f e784f 36
sc2f e741f 37 sc2f e814f 44 sc2f fell off 2 45 sc2f fell off 1 46
sc2f e781f 47 sc1f e732f 48 sc1f e730f 49 sc1f e739f 50 human
control 51 sc1f e731f 52 sc1f e812f 53 sc1f e832f 54 sc2f e778f 64
30vc2m e697m 65 30vc2m e698m 66 30vc2m e770m 67 30vc2m fell off 68
30vc2m e695m 69 30vc1m e683m 70 human control 71 30vc1m e684m 72
30vc1m fell off 73 30vc1m e693m 74 30vc1m e727m 75 60vc2m e782m 76
60vc2m fell off 2 77 60vc2m fell off 1 78 60vc2m e809m 79 60vc2m
e738m 84 60vc1m e811m 85 60vc1m e736m 86 60vc1m fell off 87 60vc1m
e734m 88 60vc1m e740m 89 sc2m fell off 2 90 human control 91 sc2m
e724m 92 sc2m e605m 93 sc2m e702m 94 sc2m fell off 1 95 sc1m e705m
96 sc1m e718m 97 sc1m e701m 98 sc1m e703m 99 sc1m e596m
INDUSTRIAL APPLICATION
[0065] Crossbreeding a dual transgenic mouse to produce a human
Lp(a) and not produce vitamin C due to lack of GULO (GULO-/-) gene
using transgenic mouse having a first knockout strain, a second
strain to make a third strain and using the first knockout strain
and fourth strain to make a fifth strain, using the third strain
and the fifth strain to make a dual transgenic mouse. Treating the
dual transgenic mouse with a Lp(a)-modulating compounds in order to
identify preventive and/or therapeutic approaches for a human
Lp(a)-related diseases. The human Lp(a)-related disease is
cardiovascular, inflammatory, infectious or degenerative in nature.
Sequence CWU 1
1
12130DNAMouse 1ctagtgtagt ctaggtgata aggatcaact 30225DNAMouse
2cagctcagag agagaatgaa tcaca 25320DNAMouse 3ctgacatccc ttaggagttc
20420DNAMouse 4agatgtgttc caggctgcaa 20519DNAMouse 5cacacactgc
agggtgaca 19616DNAMouse 6ctgcctgggt gttatc 16725DNAHuman
7cactacattt tgtgccagag atgga 25820DNAHuman 8ccctgtcctg aggctcctta
20916DNAHuman 9tcagcagccc tcttcc 161024DNAHuman 10aggtttaact
cctcctacct ccaa 241121DNAHuman 11tgagggagag ggttccatct t
211221DNAHuman 12accagataac aggaagatat g 21
* * * * *