JP2002000122A - Human lpl transduced transgenic rabbit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transgenic rabbit as a model for research of lipid metabolism and arteriosclerosis, which can excessively develop human LPL by transducing a human LPL gene to a mouse sensitive to a choloesterol added feed and apt to develop arteriosclerosis within several months. SOLUTION: The transgenic rabbit is prepared as follows: fertilized eggs are taken out from the oviducts of a donor rabbit which has been subjected to a superovulation treatment; a DNA construct containing a human LPL cDNA which is expression induced by a tri-β-actin promotor or a human macrophage receptor promotor is microinjected into the fertilized eggs under an inverted differential interference microscope; the fertilized eggs into which the DNA construct has been transduced are selected; and they are transplanted into the oviducts of a recipient female rabbit which is in a false pregnancy.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a transgenic rabbit capable of expressing human lipoprotein lipase (LPL), and more particularly, to a hyperlipidemia capable of expressing human LPL into which a human LPL gene has been introduced. Transgenic rabbits useful for the study of disease and atherosclerosis.

[0002]

2. Description of the Related Art Atherosclerosis and its complications account for about half of the causes of death in industrialized nations including Japan, and as the population ages, the socio-economic situation is now increasing. Is also the most important issue. Many hypotheses have been proposed for the cause and pathogenesis of this arteriosclerosis, but at present, the causes (risk factors) of arteriosclerosis are multiple, and if they are caused by genetic or environmental factors alone or in combination. It is considered. Among the many risk factors, the most prominent are genetic predisposition to lipoprotein metabolism and dietary hyperlipidemia.

[0003] The metabolism of blood lipoprotein, which is a complex of lipid and protein, in which lipids such as cholesterol, neutral fat (triglyceride), and phospholipid are associated with apolipoprotein, is an extrinsic pathway for processing dietary lipid. Lipoproteins are found in the blood and are affected by enzymes in both cases through an endogenous pathway that metabolizes lipids synthesized in the liver. Among them, lipoprotein lipase (LPL), which is a glycoprotein secreted by various tissues, mainly muscle and parenchymal tissues of fat, is a lipoprotein rich in neutral fat in blood by binding to vascular endothelial cells. It governs the hydrolysis of chylomicrons and triglyceride (TG) of very low density lipoprotein (VLDL). Furthermore, involvement in the metabolism of remnant lipoprotein and high density lipoprotein (HDL) is also assumed. The fatty acids released then bind to the albumin and are transferred to the parenchymal cells where they are used as an energy source or stored.

[0004] LPL (particularly LPL secreted from macrophages infiltrating the arterial wall) is involved in the binding of lipoprotein to cell surface receptors, and is presumed to play a role in the development of arteriosclerosis. Have been. Zilversmit first proposed that LPL plays an important role in arteriosclerosis because LPL promotes the formation of arterial remnant lipoproteins, which increase the deposition of blood cholesterol and triglycerides in the arterial wall. Lindqvist
Have shown that LPL hydrolysis of high triglyceride lipoproteins is important for macrophages to take up lipoproteins. In addition, LPL may bind to cell surface heparin proteoglycan sulfate and increase cellular uptake of lipoprotein microparticles (Arterioscler T
hromb Vasc Biol 1995; 15: 551-561). Because of the presence of LPL in atherosclerotic lesions,
Has been speculated (Acta Histochem Cytochem 19
98; 31: 485-492, J Lipid Res 1987; 28: 437-445, J Clin
Invest 1993; 92: 1759-1765, J Clin Invest 1992; 89: 1
544-1550, Proc Natl Acad Sci USA 1991; 88: 10143-101
47).

[0005] To study the function of LPL, five groups have made transgenic mice expressing human LPL, and three of them have systematically produced LPL.
Of transgenic mice expressing E. coli (J Biol
Chem 1994; 269: 11417-11424, J Biol Chem 1993; 268: 1
7924-17929, J Biol Chem 1994; 269: 18757-18766), 2
The group has produced transgenic mice that express LPL in specific muscles (Am J Physiol 1997; 273:
R683-R689, J Clin Invest 1995; 96: 976-986). Also,
Shimada et al. Reported that human L-transgenic mice lack human low-density lipoprotein (LDL) receptors.
We show that PL overexpression reduced dietary atherosclerosis, speculating that LPL plays a protective role against dietary atherosclerosis.

[0006] Mice are small animals, and some differences in lipid metabolism from humans have been pointed out. The major lipoprotein in serum is high-density lipoprotein (HDL) because the activity of cholesterol ester transfer protein (CETP) is lower in mice than in humans. Apoprotein B48, a constituent of chylomicron, is synthesized only in the small intestine in humans and rabbits, but is also produced in liver in mice. In addition, remnants and HDL
There is also a difference in the localization pattern of hepatic lipase, which is an important enzyme in the metabolism of hepatocytes. Humans and rabbits bind to hepatocytes and endothelial cell surface glycoproteins. It is released into the blood without binding to the surface and is in a free state. Against this background, administration of a cholesterol-supplemented diet in mice is generally not likely to cause an increase in serum cholesterol levels, as well as an atherosclerotic lesion. Furthermore, the small size of the mouse makes it very difficult to observe or quantitatively analyze atherosclerotic lesions even in the aorta. In contrast, rabbit lipoprotein constituent fractions are similar to humans and are sensitive to cholesterol and cause arteriosclerosis in a short period of time. For these reasons, human liver lipase (Fan et al., Proc. Natl. Acad. Sci. USA 1994; 9
1: 8724-8728), apolipoprotein B-100 (Fan et al.,
Arterioscl.Thromb.Vasc.Biol. 1995; 15: 1889-1899),
Apo B mRNA editing protein (Proc Natl Acad S
ci USA 1995; 92: 8483-8487), Apo E (Arterioscler T
hromb Vasc Biol 1998; 15: 1889-1899), lecithin-cholesterol acyltransferase (J Biol Chem 19
96; 271: 4396-4402) and ApoA-I (Arterioscler Thr)
A number of transgenic rabbit models have been generated, such as omb Vasc Biol 1996; 16: 1424-1429).

[0007]

However, there are still many unclear points as to how LPL is specifically involved in atherosclerosis, and particularly secretion from macrophage-derived foam cells infiltrating atherosclerotic lesions. The role of LPL is completely unknown, and it is impossible to analyze it with existing animal models. Therefore, the present inventors have attempted to produce a transgenic rabbit expressing human LPL with the aim of developing a research model for elucidating the role of LPL in arteriosclerosis and lipoprotein metabolism. An object of the present invention is to introduce a human LPL gene into rabbits that are susceptible to a cholesterol-added diet and easily develop arteriosclerosis in a few months, whereby human LPL can be overexpressed, resulting in abnormal lipid metabolism, particularly hyperlipidemia. To provide a model transgenic rabbit for the study of atherosclerosis and atherosclerosis.

[0008]

Means for Solving the Problems The production of transgenic rabbits requires a higher level of technology and breeding environment than the production of transgenic mice, and even if these conditions are established, it is more successful than mice. At present, the percentage of positive transgenic rabbits is very low. Specially, it is necessary to develop basic technologies such as production of DNA constructs with high purity and high expression efficiency, egg collection technology by hormone administration, etc., technology to make pregnancy with high probability, or improvement of division ability of embryos injected with genes. Ingenuity and technology development are required. Therefore, the present inventors have developed a transgenic rabbit production technique (Fan et al., Pathology Internat) developed by the present inventors using a large number of rabbits.
ional 1999; 49: 583-594), and the presence and expression of the transgene were confirmed by Southern blotting and Northern blotting, respectively, and a model transgenic rabbit overexpressing human LPL was prepared. Have been found, and the present invention has been completed.

That is, the present invention relates to a DNA construct containing a DNA encoding human lipoprotein lipase.
A transgenic rabbit characterized by expressing human lipoprotein lipase (Claim 1), a transgenic rabbit characterized by expressing human lipoprotein lipase systemically (Claim 2), DNA
3. The transgenic rabbit according to claim 1 or 2, wherein the construct comprises a CMV-IE enhancer, an avian β-actin promoter, a rabbit β-globin intron, and a human lipoprotein lipase cDNA. The transgenic rabbit L17 which expresses lipase systemically (Claim 4) or the transgenic rabbit which expresses human lipoprotein lipase in a macrophage-specific manner (Claim 5).
And the DNA construct comprises a human macrophage scavenger receptor enhancer / promoter and a human lipoprotein lipase cDNA.
The transgenic rabbit according to claim 6 (claim 6), the transgenic rabbit L14 that expresses human lipoprotein lipase in a macrophage-specific manner (claim 7), and the transgenic rabbit according to any one of claims 1 to 7,
Administering a test substance and measuring / evaluating a change in the expression of human LPL in the transgenic rabbit, wherein the agent is a prophylactic and / or ameliorating agent for a disease caused by excessive or low expression of human LPL. (Claim 8).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As the transgenic rabbit of the present invention, DN containing DNA encoding human LPL is used.
There are no particular restrictions on the rabbit strain into which the A construct has been introduced and which can express human LPL. (1) CMV-IE enhancer, avian β-actin promoter, rabbit β-globin intron, human liposome A transgenic rabbit capable of systemically expressing human LPL into which a DNA construct containing a protein lipase cDNA has been introduced, or (2) a DNA construct containing a human macrophage scavenger receptor enhancer / promoter and a human lipoprotein lipase cDNA. Transgenic rabbits capable of expressing human LPL in a macrophage-specific manner can be specifically mentioned. These transgenic rabbits have been passaged in the Pathology Laboratory at the University of Tsukuba, and under certain conditions, persons concerned can receive a subdivision.

Transgenic rabbits capable of expressing human LPL systemically are useful for studying lipoprotein metabolism and abnormalities, as well as arteriosclerosis and lifestyle-related diseases. In particular, transgenic rabbits capable of specifically expressing human LPL in macrophages are useful for studying the role of LPL secreted from macrophage-derived foam cells infiltrating atherosclerotic lesions. Can contribute to elucidation of the etiology and mechanism of arteriosclerosis, which is related to about half of the deaths in Japanese.

The method of establishing the transgenic rabbit of the present invention includes the method reported by the present inventors (Fan et al., Proc. Natl. Acad. Sci. USA 1994; 91: 8724).
-8728, Fan et al., Arterioscl. Thromb. Vasc. Biol. 199
5; 15: 1889-1899). And
The DNA construct used for gene transfer preferably contains a CMV-IE enhancer, an avian β-actin promoter, a rabbit β-globin intron, and a human LPL cDNA. Although the promoter for expressing the human LPL gene is not particularly limited, for example, avian β-actin promoter, human CMV promoter,
A human macrophage scavenger receptor promoter,
The mouse von Willebrand factor promoter can be used, but the avian β-actin promoter is preferred because the human LPL gene is expressed systemically in various rabbit organs, and the human macrophage scavenger is used for specific expression in macrophages. Preferably, a receptor promoter is used. In the above DNA constructs, human L, particularly using the avian β-actin promoter
When regulating the expression of the PL gene, human LPL cDNA
The expression efficiency can be increased by inserting a rabbit β-globin intron in the upstream region of E. coli. As a method for introducing a DNA construct containing human LPL cDNA into a fertilized egg, a nuclear transfer method and the like that we are currently developing can be used instead of the microinjection method.

The transgenic rabbit of the present invention can be advantageously used to elucidate the mechanisms of lipid metabolism and arteriosclerosis, and can overexpress human LPL. It can be used for screening for a preventive drug or symptom ameliorating agent for a disease or symptom caused by the disease, for example, dietary hyperlipidemia or diabetes. Such screening methods include:
A test substance is administered to the transgenic rabbit of the present invention, and human LPL in the transgenic rabbit is administered.
A method for measuring and evaluating a change in expression of is mentioned. In addition, such a measurement / evaluation method includes human LPL.
Quantification of human LPL protein by ELISA using an antibody that specifically recognizes only lipoprotein, separation and quantification of lipoprotein constituent fractions from rabbit plasma by gel filtration column chromatography, fractional ultracentrifugation, etc. These methods make it possible to evaluate the effect of a test substance on human LPL in a living body. And these measurements
At the time of evaluation, it is preferable to use the human LPL overexpressing transgenic rabbit of the present invention and a wild-type rabbit of the same species simultaneously, since an accurate comparative experiment can be performed at the individual level.

[0014]

The present invention will be described below with reference to examples.
The present invention is not limited by such embodiments. Example 1 [Transgenic rabbit overexpressing human LPL systemically] (Preparation of transgenic rabbit) Transgenic rabbits are described in the literature (Fan et al., Proc. N).
atl.Acad.Sci. USA 1994; 91: 8724-8728, Hammer et a
l, 1985, Taylor and Fan, 1997). Japanese white rabbit (manufactured by Tokyo Laboratory Animal Co., Ltd .; manufactured by Japan SLC Co.) 40 which is not contaminated by a specific pathogen
45 birds were used. On the first day, donor rabbits (4 to 6 months old) were intraovulated by intramuscular injection of 150 IU of pregnant horse serum (Sigma). On the fourth day, 150 IU of human chorionic gonadotropin (manufactured by Sigma) was injected intravenously with the donor rabbit together with a fertile male rabbit, and 17 to 19 hours later, the fertilized egg was tubalized at the University of Tsukuba Animal Center. Removed from

CMV-IE enhancer, avian β-actin promoter (Niwa et al., Gene, 1991; 108: 193-200), rabbit β-globin intron, human LPL cDNA (W
ion et al., Science, 1987; 235: 1638-1641), a DNA construct of about 3.8 kb (see FIG. 1) was prepared at 10 mM.
Suspended in a buffer containing Tris (pH 7.5) and 0.25 mM EDTA to a concentration of 4 to 8 ng / μl,
The embryos were microinjected under 200-fold magnification using an inverted differential interference microscope (“IX70” manufactured by Olympus Corporation). Oocytes into which the DNA construct had been introduced were selected, and 15 to 30 eggs per chick were transplanted to the margin of the oviduct of the mother of 116 pseudopregnant recipient female rabbits, and 166 pups were obtained. A rabbit was born.

(Detection of Foreign Transgene) In order to isolate genomic DNA, a biopsy tissue was excised from a rabbit ear of 2-3 weeks after birth, and digested with a digestion buffer (50 mM Tris-HC).
1, 0.5 mM SDS containing 100 mM EDTA, 200 μg / ml proteinase K) at 55 ° C. overnight, phenol: chloroform: isoamyl alcohol (25: 24: 1 vol / vol) (Life Techn.
The extraction and purification were carried out using “Gibco BRL” manufactured by ology. 1.58 kb human LPL cD obtained by digesting plasmid pCAL-1-hLPL with EcoRI
Using a NA-labeled probe, the foreign transgene was detected using Southern blotting. 10 μg of genomic DNA digested with the EcoRI restriction enzyme was electrophoresed on a 1% agarose gel, and subjected to a turbo blotter system (Schl).
eicher and Schunell). The genomic fragment immobilized on the membrane is hybridized with a ³² P-labeled cDNA probe synthesized by Prime-It II random primer labeling kit (manufactured by Stratagene), and the foreign introduced gene is subjected to autoradiography. The presence or absence of expression was detected.

Seven out of 166 birds were found to be transgenic rabbits expressing a foreign transgene by Southern blotting (see FIG. 2). The total positive rate of transgenic rabbits in the selected pups was 3.6%, which reflects the difficulty of producing transgenic rabbits compared to mice. All three surviving transgenic founder rabbits were male (see Table 1). Of which L
01 is infertile due to spray leg (Lindsey and Fox, 1994), L04 is mosaic and the foreign gene is not inherited, but L17 has normal fertility and is systematized. This transgenic rabbit L17 has been passaged in the Pathology Laboratory at the University of Tsukuba, and medical personnel can receive it under certain conditions.

[0018]

[Table 1]

(Separation and analysis of RNA) m of human LPL
RNA distribution was examined using Northern blotting. Various tissues of the transgenic rabbit (aorta,
Total RNA was isolated from heart, lung, kidney, adrenal gland, liver, spleen, intestine, stomach, muscle, thymus, brain, bone marrow, testis, epididymis. 10 μg of RNA denatured in the presence of dimethyl sulfoxide and glyoxal was subjected to 1.2% agarose gel electrophoresis, then transferred to a nylon membrane, and subjected to hybridization using a ³² P-labeled human LPL cDNA probe. As internal standard the same amount of ³² P labeled human β- actin probe (ClontechLaboratorie
s company). FIG. 3 shows the results of the Northern blot. From FIG. 3, human LPL is composed of multiple tissues, mainly kidneys,
It was found to be expressed in the adrenal gland and intestine. This human LPL
The tissue distribution of expression was similar to that of transgenic mice generated in the same way (Shimada et al.,
1993).

(In situ hybridization and immunohistochemical staining) Human LPL was analyzed by Northern blot.
Was found to be expressed mainly in the kidney, adrenal gland and intestine. To identify cells expressing LPL in the intestine, Prime-It random primer labeling kit (Challah et al., Cardiovasc Res., 1995; 30: 2
31-239) ³⁵ L-labeled human LPL synthesized using
In situ hybridization was performed using a cDNA probe. All samples were excised from the tissue, polyvinyl alcohol 10.24%, 4.26% of polyethylene glycol, a non-reactive components 85.5% (w / w) containing Tissue-Tek ^R O. C. T. Embedded in compound and frozen in liquid nitrogen. Frozen sections were prepared and prepared with 4% paraformaldehyde and stored at -20 ° C until use.

The sections were subjected to a hybridization solution (50% formamide, 4 × SSC, 10% dextran sulfate, salmon sperm DNA 250 μg / ml, herring DNA 250 μg / ml, yeast tRNA 400 μg / m 2).
l, and 0.5 × 10 ⁶ cp of a ³⁵ S-labeled human LPL cDNA probe diluted with 0.5 times Denhardt's solution).
and 18 hours at 37 ° C.
The sample was then rinsed at 45 ° C. using 4 × SSC and 40% formamide (2 × 15 min), followed by 4 × SS
Rinse at 60 ° C. using C for 30 minutes (30 minutes), use 1 × SSC at room temperature (3 times for 15 minutes), and use 0.5 × SSC at room temperature (2 times for 30 minutes). And dehydrated with ethanol. Slide the radiographic emulsion NR
-M2 (made by Konica Co.), 4 ° C for 2-4 weeks
Exposed. The sections were then developed and fixed and counterstained with hematoxylin and eosin. As a negative control, RNAs were used before hybridization with the cDNA probe.
Serial sections treated at 50 μg / ml were used. To further confirm LPL protein expression in these tissues, an anti-human LPL monoclonal mouse antibody 5 provided by Dr. J. Brunzell of the University of Washington, Seattle 5
Immunohistochemical staining was performed using D2. FIG. 4 shows the results. FIG. 4 shows that human LPL protein (A) and mRNA
(C) was found to be present in the intestinal epithelial cells of both transgenic rabbits. (B, D) in FIG. 4 shows a negative control in the intestine of a normal rabbit. The magnification is 66 times for A and B, and 80 times for C and D.

(Analysis of LPL Protein and Enzyme Activity) Transgenic rabbits and normal rabbits were each injected with 30 units of heparin mass per kg of body weight, and 10 minutes later, post-heparin plasma was collected. Collected. LP to measure LPL protein
Two anti-human LPLs that recognize different epitopes on L
ELISA using a combination of monoclonal antibodies
Was done. In addition, the enzymatic action of LPL was measured using a ¹⁴ C-labeled trioleinfosfatidylcholine emulsion substrate (Bensadoun et al., J Biol Chem, 1974; 249: 2).
220-2227). As shown in Table 2, transgenic rabbits had LPL activity approximately three times higher than normal rabbits.

[0023]

[Table 2]

Example 2 [Transgenic rabbit overexpressing human LPL specifically in macrophages] Japanese white rabbit (manufactured by Tokyo Laboratory Animal Company; Japan S)
Using 1114 birds (manufactured by LC Inc.), the DN of Example 1 was used.
The macrophage scavenger receptor promoter was used as shown in FIG. 1 to replace the avian β-actin promoter and the like in the A construct.
LPL transgenic rabbits that specifically overexpressed only in macrophages were prepared. Six out of 29 born birds were transgenic founders, but two surviving rabbits (see Table 1). FIG. 5 shows the results of Southern blot and Northern blot, which confirmed the presence of the foreign transgene and the expression of mRNA in the transgenic founder, respectively. Currently living L14 has normal fertility and is systematized. This transgenic rabbit L14 has been passaged in the Pathology Laboratory of the University of Tsukuba, and related persons can be obtained under certain conditions.

[0025]

According to the present invention, transgenic rabbits overexpressing human LPL were successfully produced, and transgenic rabbits useful as a model for studying lipid metabolism and atherosclerosis can be obtained. WHHL rabbits isolated by genetic methods in 1980 have been widely used as model animals for studying lipid metabolism and atherosclerosis, but no other useful rabbit model is known. By applying the present invention, transgenic rabbits using different exogenous transgenes can be produced, and the possibility of analyzing the causes and pathogenesis of atherosclerosis in vivo, which was impossible in the past, can be opened up. .

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a DNA construct used for producing the transgenic rabbits of Examples 1 and 2.

FIG. 2 shows the results of Southern blot of the genome in the transgenic rabbit of Example 1.

FIG. 3 shows various tissues of the transgenic rabbit of Example 1 (aorta, heart, lung, kidney, adrenal gland, liver, spleen,
It is a figure which shows the result of Northern blot in intestine, stomach, muscle, thymus, brain, bone marrow, testis, epididymis).

FIG. 4 shows the results of immunohistochemical staining of the transgenic rabbit of Example 1.

FIG. 5 shows the results of Southern blot of the genome in the transgenic rabbit of Example 2 and Northern blot of macrophages (peritoneal macrophage, alveolar macrophage).

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Claims (8)

[Claims] 1. A DNA encoding human lipoprotein lipase
A transgenic rabbit into which a DNA construct containing A has been introduced and which expresses human lipoprotein lipase. 2. The transgenic rabbit according to claim 1, which expresses human lipoprotein lipase systemically. 3. The method of claim 1, wherein the DNA construct is a CMV-IE enhancer,
The transgenic rabbit according to claim 1 or 2, comprising avian β-actin promoter, rabbit β-globin intron, and human lipoprotein lipase cDNA. 4. A transgenic rabbit L17 that systemically expresses human lipoprotein lipase. 5. The transgenic rabbit according to claim 1, which expresses human lipoprotein lipase in a macrophage-specific manner. 6. The transgenic rabbit according to claim 1, wherein the DNA construct comprises a human macrophage scavenger receptor enhancer / promoter and a human lipoprotein lipase cDNA. 7. A transgenic rabbit L14 that expresses human lipoprotein lipase in a macrophage-specific manner. 8. A human LPL, which comprises administering a test substance to the transgenic rabbit according to claim 1 and measuring / evaluating a change in the expression of human LPL in said transgenic rabbit. A method for screening a preventive and / or symptom-ameliorating agent for a disease caused by over- or low-expression of a.