JPH0491731A - Hla-bw 52 gene-converted non-human mammal - Google Patents

Abstract

PURPOSE:To obtain a gene-converted non-human mammal having HLA-Bw 52 gene by introducing a reproductive cell and a somatic cell into a non-human mammal or a progenitor of a non-human mammal at the initial stage of development. CONSTITUTION:A gene-converted non-human mammal can be produced either by introducing HLA-Bw 52 gene into a fertilized ovum or an early embryo of a non-human mammal, transplanting the fertilized ovum or the early embryo into the uterus of a temporary mother and letting the mother to deliver a child or by introducing HLA-Bw 52 gene into a germinal stem cell, transplanting the stem cell together with an embryo of a non-human mammal into the uterus of a temporary mother and letting the mother to deliver a chimera child. The reproductive cell of the chimera child contains HLA-Bw 52 gene while the somatic cell does not contain the gene. Accordingly, the chimera child is subcultured in order to introduce the HLA-Bw 52 gene into both reproductive cell and somatic cell. The animal of the next generation and thereafter is a gene- converted non-human mammal.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to HLA (hu+xan 1 eukocyte
Transgenic non-human mammals (transgenic non-humans) that have introduced the
an mamlals).

[Conventional technology]

During human organ transplantation, HLA antigens play an important role in transplant immunity as antigens that determine whether or not the graft will take hold.

HLA antigens are also called major histocompatibility antigens because they are closely related to the rejection phenomenon in transplant immunity, and the gene group encoding them is called the major histocompatibility complex (MHC).

HLA is human MHC, and HLA class I antigens are membrane proteins present on cell membranes, including A, B, and C.
It is controlled by three genetic loci. Class I antigen is a glycoprotein (α chain) with a molecular weight of 44 kd and a molecular weight of 12 kcl.
It has a structure non-covalently associated with protein V (β2-microglobulin) of α1. α2. There are three domains of α3, followed by an intracellularly embedded region (transtse
mbrane Region) and regions located within the cell (cytoplasm*ic domain) are lined up. β2-microglobulin consists of one domain and is linked to the α chain.

HLA class ■ Antigen is membrane Wi! D as an antigen is a heterodimer consisting of a non-covalent bond of the alpha chain with a molecular weight of 35 kd and the beta chain with a molecular weight of 27 kd, which are the sex proteins.
There are P antigen, DQ antigen, and DR antigen, but there are slight differences in the molecular sizes of their constituent chains.

The HLA gene region exists on the short arm of human chromosome 6 over a distance of 2.5 centimorgans (cM), from the centromere side to the class ■ region (1, OcM) and the class ■ region (between DR and B). -〇, 7cM), class r Si region (0
, ScM).

(Jordan, B. R., et al.
, Rev, , 84, 73. One of the methods for testing HLA antigens is a serological method.

As a serological method, the complement-dependent cytotoxicity test (T
erasaki, P, 1. ancl McClella
nd, J., and D.

Nature (London), 204. pp9
9B-1000, 1964) is the standard method. This method uses alloantiserum and uses rabbit serum as complement to detect lymphocyte cytotoxicity (lymphocytotoxicity).
), but the antiserum is an alloserum from a pregnant woman with multiple births, and the antiserum exhibits cross-reactivity (croSs reaction). Furthermore, since antiserum is obtained from humans, a stable supply of reproducible serum is impossible, and anti-blood/+1 against alloantigens, which are expressed in low amounts, cannot be obtained, so monoclonal antibodies are recommended as an alternative to alloserum. development is desired. 198
At the 4th International Miw & Conformity Workshop, approx.
Fifty types of monoclonal antibodies have been submitted. (Fa
uchet, R., Bonder, J.G., Ken
nedy, L. J. et al.: HLA-
AB, Cll1onoclonal antibo
dies, Histocompatibility
Testing 1984 (Albert, E. D.,
Baur, M.P.

Mayr J, R, ed.), Springer-Ve.
rlag, Berlin, Heidelberg, Ne
w York, Tokyo: 211, 1984) Anti-HL
Monoclonal antibody A can generally be produced by fusing lung cells obtained by immunizing a mouse with human lymphocytes and mouse myeloma cells. (O4, V,
T., Herzenberg disturbance, A.: Immunog.
lobulin-Producing hybrid
cell 1ines, 5 selected IIet
hods in Im+nuno1ogy, 351.1
981) However, to date, among the monoclonal antibodies that have been subjected to HLA typing, for example, monoclonal antibodies against allotypes of HLA-B antigen include Bw4, Bw6. B7 B8. B1
3. Currently, only monoclonal antibodies that monospecifically bind to B27 have been obtained, and the development of monoclonal antibodies that have monospecificity to many other allotypes is expected. Furthermore, there are many alloantigens for which even reactive monoclonal antibodies have not yet been obtained. For such alloantigens, monoclonal antibodies that have cross-reactivity with other alloantigens are also effective. Recently, further improvements have been made in methods for producing anti-HLA monoclonal antibodies. In the antibody production method using peripheral lymphocytes, which are dominated by human T lymphocytes, as an immunogen, there are many human-derived antigens, so this method is effective against many antigen sites other than the target HLA allotype antigen. This method has improved the ability to obtain monoclonal antibodies that show reactivity, and transforms cells by introducing a human HLA antigen gene clone into eukaryotic cells derived from an immunized animal and administering the transformed cells to the immunized animal. This is a method for obtaining lung cells that efficiently produce monoclonal antibodies against the human (LA) antigen expressed above.
dies to HL A-DP-transfe
cted mouseL cell:Proc, Nat
l, Acad, Sci,,ll5A,33. pp 3
4173421.1986) However, species-specific antigenic determinants (isotypes) that are not recognized between humans are more important than antigenic determinants (allotypes) that have minute differences that can be recognized between humans. This method also has the problem of producing many monoclonal antibodies directed against the isotype, that is, monoclonal antibodies that cross-react with multiple alloantigens, since the warmer version has stronger antigenicity for mice.

transgenic animal
s) is produced by introducing a foreign gene. Many transgenic mice have been created using mice because they are easy to conduct experiments and are easy to handle.

The characteristics of transgenic animals are that the introduced gene is integrated into normal cells, and the fertilized egg into which the gene has been introduced undergoes developmental differentiation and becomes a single individual, so the foreign gene is transferred to all cells that make up the individual. It may be integrated at the same location on the chromosome of the

Transgenic mice have been applied to the following themes:

(1) Analysis of the relationship between gene structure and expression (Kondoh
H, et al. (1987) Dev, Biol, 12
0. ppI77-185) (Brinster, Fl.
L, etal, (1983) Nature 306+
pp(2) Creation of human disease model mice (Wakas
ugi s.

et al, :A transgenic mouse
Nodel of familiar amyloid
polyneuropathy, Proc Jpn
-Acad 63:344, 1987) (Leder
et al, :TRANSGENICNONHUMAN
MAMMALS, tlnitedStatePaten
tNo.

4.736,866 (1988) ) (3) Analysis of the role of gene products within cells or individuals. In the field of immunity, transgenics are used to analyze the role of MHC class I and class II antigens.

Kumouse is used.

(Frels, h, 1. et al, :5science
, 228:577.1985) (Le Meur, ?
1. et al.: Nature, 3] 6:38.1
985) (YamanuraJ, et al.: Nat.
ure 316:67.1985) (1e to Femia
Vits et al.: Nature 329: pp.
447449.1987) Frels et al. developed swine class I (SLA) transgenic mice, Le Meur et al. developed mouse E transgenic mice, Yamasura et al.
et al. produced mousebill transgenic mice, Femi
a Kievits et al. have generated HLA-827 transgenic mice.

Although not yet publicly known, a method for producing anti-HLA monoclonal antibodies using genetically modified animals has been developed in a recent application filed by the applicant (Japanese Patent Application No. 2-26828).

This method uses an HLA gene-transformed animal different from the immunization source as the immunized animal used when immunizing with HLA alloantigens. This is a method for efficiently producing monoclonal antibodies.

There are still only a few types of transgenic animals that have introduced HLA genes, and in order to have the various usefulness mentioned above, HL animals that have introduced genes encoding various HLA alloantigens have been developed.
A: The development of transgenic animals is desired.

[Problem to be solved by the invention]

The purpose of the present invention is to obtain an HLA-Bw52 transgenic animal, which has not been obtained in the past.

[Means and Effects for Solving the Problems] The present invention provides HLA-Bw whose germ cells and somatic cells are introduced into a non-human mammal or an ancestor of this non-human mammal at an early stage of ordering.
It is a transgenic non-human mammal with 52 genes.

Examples of non-human mammals that can be used include primates such as rhesus monkeys and chimpanzees, and rodents such as mice and rats.

As a method for obtaining gene-transformed non-human mammals, HLA-Bw5 is added to fertilized eggs or early embryos of non-human mammals.
2. After introducing the 2 genes, the fertilized eggs or early-stage embryos are implanted into the uterus of a foster mother and the foster mother gives birth to an individual, and the embryonic stem cells that have introduced the HLA Bw52 gene are used as embryos of non-human mammals. There is a method of transplanting them together into the uterus of a foster mother and causing the foster mother to give birth to a chimeric individual.

The germ cells of this chimera individual have the HLA-Bw52 gene, but the somatic cells do not have the HLA-Bw52 gene, so this chimera individual was subcultured so that both the germ cells and somatic cells had the HLA-Bw52 gene. Genetically transformed non-human mammals can be obtained from individuals of subsequent generations.

Methods for introducing HLA genes into fertilized eggs, early embryos, and embryonic stem cells include (1) directly injecting isolated HLA genes into cells using a micromanipulator; (2) injecting HLA genes into cells using a micromanipulator; In addition to the method of infecting cells with the integrated recombinant retrovirus and the method of (3) treating with high concentration calcium chloride to incorporate HLA genes into cells, other known methods can be used.

〔Example] The present invention will be explained based on examples.

HLA-Bw52tonesgeni.kumouse HLA
- Purification of Bw52 gene 6.4 kb of HLA-Bw52 gene having the base sequence shown in Fig. 1 was converted into EcoRI 5ite T: PBR
Plasmid PHLA-Bw52 contained in 328 3
0 μg was reacted with 90 units of EcoRT at 37°C for 60 minutes to cleave the gene. The new cut piece was added to the end of an agarose gel and subjected to electrophoresis, and the 6.4 kb band was cut out with a knife to separate the HLA and -Bw52 genes.

The isolated gene was added to approximately twice the amount of 5 odiulIliodi.
Add 1.5+d to the test tube along with the DE solution and dissolve in a 55°C water bath. Next, add 5-10μ to the test tube.
silica matrix (GIASSMILK, G
(manufactured by ENECLEANTM), mixed, and incubated at °C. After centrifuging the test tube at 1500 rpm for 5 seconds, discard the supernatant. 10n+MTris pH7
, 5, 1 mM EDTAO, IM NaCl, and 0.2 d of a washing solution consisting of 50% ethanol is added to the test tube, stirred with a pipette, and then centrifuged at 15,000 rpm for 5 seconds.

This washing operation is performed three times. After centrifugation, remove the supernatant and add 5QM Tris pH 9,0, 0.2 FIN to the test tube.
Add 20 μffi of aCI solution and incubate at 60°C for 30 minutes. test tube at 15000 rpm 30
fJ: Centrifuge for a while and transfer the supernatant to a new test tube. Add 50 nM Tris pH 9, O, 0 to the test tube containing this supernatant.
, 2M NaCl solution at 20 μC and incubation at 60°C for 30 minutes, followed by centrifugation at 15,000 rpm for 30 seconds to collect the supernatant.

Ethanol is added to this supernatant to precipitate the DNA. D
After drying the NA precipitate, 10mM Tris pH7,
5. Dissolve in 0.5mM EDTA solution to prepare a concentration of 500 copies/pi.

Collection of fertilized eggs A C3H female mouse (C3H/lle; purchased from Shizuoka Experimental Animal Center) was intraperitoneally injected with 5 units of pregnant mare serum (PMS), and 48 hours later, 5 units of chorionic gonadotropin (HCG) was injected into C3Ha. Put it in the tribe gauge where the mouse is. -Female mice are observed in the evening and mating is confirmed the next morning, and then the oviducts and part of the uterus are removed. Under a stereomicroscope, the ampulla of the oviduct is cut open in a petri dish containing a culture solution containing hyaluronidase 11Ig/- to remove fertilized eggs.

After washing the fertilized eggs treated with hyaluronidase three times with culture solution, they are placed in a carbon dioxide incubator at 37° C. and cultured.

A holding pipette for fixing a fertilized egg and an injection pipette for injecting a DNA solution are connected to the gene introduction micromanipulator. Make a drop of culture solution and a drop of DNA solution in a culture dish, and pour paraffin oil over the top to cover it. Transfer the collected fertilized eggs to a drop of culture solution, and set the culture date on the injection microscope. DNA with injection pipette
Aspirate the solution. With the fertilized egg fixed with a holding pipette, a DNA solution is injected into the male pronucleus of the fertilized egg using an injection pipette.

Transplantation of gene-transferred embryos into foster mothers Perform vasectomy on male mice to make them infertile. With this male Matsusu! ICR mice are mated and kept in a pseudopregnant state. 6
mg/R12 Mbutal per southwest 0,3 url
Anesthetize by injecting intraperitoneally. Next, the mouse is placed face down and the skin and substratum are incised from the dorsal side. Pinch the fatty mass around the ovary with a Vince Soto and pull it out, find the oviduct opening, and use a transfer pipette to transfer the transgenic fertilized egg into the oviduct. After transplantation, the ovary, sinus tube, and uterus are returned to the abdominal cavity.
The partial layers are sutured and the skin is closed with crimp. Mice born about 20 days later are raised by IcR mice that have already given birth to offspring.

Confirmation of transgenic mice Is the gene transferred to mice that have grown up to about 4 weeks old?
In addition, whether or not it was expressed was confirmed using the following two methods.

1) Collect DNA from the tail and confirm whether the HLA-Bw52 gene has been integrated into the mouse chromosome using the do-no-dopro-cyto method.

Cut off 11 tails from 4-week-old mice to give 1.5Ill.
Put it in a test tube. 0.7d SE T buffer (5d
IM Tris-HCI pH8,o 50I1
10.2MEDT^, 2.5M120%SO5,42,
5 relatives H20) and 20μ! ! ,IO@g/me pro
Add teinase K to the test tube. Dissolve the tail by shaking overnight at 37°C. Transfer 70μl from the test tube to a new test tube and make 140μl! TE buffer (lomM Tris-C
Dilute by adding 1 mM EDTA pH 8,o). Furthermore, 200 μ in a new test tube! Add phenol and centrifuge at 15,000 rpm for 5 minutes. 20p of supernatant
l3M Sodium acetate, 500μ
f Transfer to another test tube with 99% ethanol, stir, and incubate at -70°C for 15 minutes. Next, 4°
After centrifugation at C15000 rpm for 15 minutes, discard the supernatant and dry. The dried pellet was treated with 2M NaCl, 0.
Add 100 μf of 1N NaOH solution and dissolve well.
After incubating at 00°C for 65 minutes, rapidly cool with ice water to prepare a sample. Nitrocellulose filter 15CI
Soak the X12.5Ω in water for 5 minutes, then in 3x SSC buffer for 5 minutes. Dot plot equipment (Bio-R
3 between the rubber gasket (manufactured by ad) and the sample plate.
A Watfun 3MM filter paper soaked in twice the amount of SSC buffer is placed, and a nitrocellulose filter is placed on top of it and fixed.

Add the sample to the dot plot device and let it absorb into the filter. Remove the nitrocellulose filter and dry it. Place the DNA in a vacuum dryer at 80°C for 2 hours.
is fixed on a nitrocellulose filter. Place this nitrocellulose filter into the hybridization hack. Prehybritization solution (50x Denhardt's 5solution O, 8d1
0+RFor+wamide, 0.2dlO% S
D S , 0.5 m of 4 / salmon sperm DNA, 6.6 paper 20x 5 SC 1,9*Hz) was added and the hang was sealed.
Incubate for 2 hours at 2°C. "Rahel with P-dCTP", X 10'cps+ HLA-Bw52
Place the DNA probe and 4 mg/0.5 of Mesake sperm DNA in a glass test tube and heat in a 100'C water bath for 5 minutes, then cool on ice. 0.81 in this glass test tube
50 times IlDenhardt's 5solution
, 10aRP.

rmamide, 0.2IRI110% S D S
, 6.6d Add a mixture of 20x SSC solution and hybridize? 8'a. Discard the brehybridization solution from the hybridization hack, refill the hybridization solution, seal the hack, and incubate at 42" CI for 8 hours.

Remove the nitrocellulose membrane from the bag and double the 5S
The nitrocellulose membrane is dried after washing with a washing solution consisting of C10, 1% SDS for 30 minutes at 37°C twice and then twice for 30 minutes at 52"C. The dried nitrocellulose membrane is fixed on a -hatman 3MM filter paper and Place the sample in a X-ray film cassette and expose it to X-ray film for 3-18 hours, then develop the film. If the HLA-Bw52 gene is present in the sample, a black dot will appear. This black dot can be confirmed with a photograph. HLA-Bw52
Determine whether the gene has been integrated into the mouse chromosome.

The gene introduction procedure described above was performed to produce 154 mice. As a result of performing the hybridization described above (1) on all mice, three of them were Bw5214-9 females. Us, B w52-21
-4 t17 mice and B w52-21-7117 mice showed positive results, confirming that the HLA-Bw52 gene had been incorporated.

Bw generated from microinjected fertilized rabbits of Tonegenic mice
52-14-9 N mouse (F, mouse) is HLA-
Since it is a Bw52 transgenic, in order to establish a transgenic line, this Bw52-14-9 mouse (female) is bred with another mouse of the same species and gives birth to a pup.

Bw52-14-9 mice (♀) were transformed into normal C3H/He
They were placed in the same cage as male mice and mated. She gave birth to several pups in about 3 weeks. This process was repeated, resulting in a total of 4 pregnancies, resulting in 19 Fl.

As a result of dot plot hybridization as described above for all F mice, B w52-F,
-2-4, Bw52-F, -41BWO2P+ 4
The Bw52 gene was inserted into three F mice of No. 7.

The embryonic stem cells into which the desired HLA gene has been introduced are combined with the blastula of a non-human mammalian mammal, grown into an individual to obtain a chimeric individual, and this chimeric individual is passaged to carry out the introduced gene in the next generation. A method for obtaining a transgenic non-human mammal having the following will be explained based on Examples.

Embryo collection C3H#? Mice (C3H/He, purchased from Shizuoka Experimental Animal Center) were intraperitoneally injected with 5 units of PMS, and 48 hours later, chorionic gonadotropin (H
CG) Inject position 5Ii. Then change this mouse to C3
Place it in the gauge containing the Hil mouse. - Observe the mice in the evening to confirm whether or not they have mated. Two days later, the mated female mice are killed and the oviducts and part of the uterus are removed. Early embryos at the 8-12 division stage are collected from the excised V& organ. These early embryos are treated with hyaluronidase, washed with culture solution, and then cultured at 37°C in a carbon dioxide incubator.

In the above operation, an early embryo at the 812 division stage was removed from a pregnant female mouse, but a blastocyst (blastula) may also be removed.

Embryonic stem cells (ES cells)
Introduction of the gene into ES cells The HLA-Bw52 gene prepared in the above example can be introduced into ES cells by the following procedure.

First, a culture solution and a drop of a solution of HLA-BW52 genomic DNA having the base sequence shown in the first step are prepared in a culture dish, and paraffin oil is poured over the drop to cover the drop. Next, the collected ES cells are transferred to a drop of culture medium. After that, place this Day 7 soybean for culture into a microscope for injection.

to This 8-page microscope is equipped with a micromanipulator, which is connected to a holding pipette for fixing cells and an injection pipette for injecting a DNA solution. . After setting the dataset on the microscope, first aspirate the DNA solution with an injection pipette. Next, with the ESWj cell fixed with a holding pipette, the DNA solution that has been aspirated into an injection pipette is injected into the nucleus of the ES cell.

Injection of transformed ES cells into embryos The embryos prepared in the above examples were pre-warmed and transferred to a cooled injection chamber.

Sufficient number of ES cells for one injection (approximately 1~
15) into the injection tube, slowly insert the injection pipette into the embryo, slowly push out the cells, and inject the ES cells.

The injected embryo is covered with DMEM oil!
Place in a 10% FC3 drop and culture at 3T'C for 1 day.

Transplantation of ES cell-injected embryos into foster parent mice First, male mice were rendered infertile by vasectomy. Next, this male mouse and @ICR mouse are mated to make the female mouse pseudopregnant.

Three days later, the embryo injected with the ES cells obtained above is implanted into this pseudopregnant female mouse as follows. First, each animal is anesthetized by intraperitoneally injecting 6 + + + g/0.3 g of Shikyo Mbutal per animal. Next, the mouse was placed face down and the skin and substratum were incised from the dorsal side.
Pinch the fat mass around the ovary with a bottle set and pull it out. The oviduct opening is found from the extracted fat mass, and the ES cell-injected embryo is transferred into the oviduct using a bipent for transfer. After the fertilized egg is implanted, the ovaries, fallopian tubes, uterus, etc. are placed back into the abdominal cavity, the partial layers are sutured, and the skin is secured with clips. Mice born about 20 days later are raised by JCR mice that have already given birth.

Confirm the transgenic mouse to determine whether the gene has been transferred to the germ cells of the mouse.

It is confirmed whether the LA-Bw52 gene has been integrated into the DNA collected from the tail of this mouse (F) using the Donoto blot hybridization method described in the above-mentioned Examples.

Next, this F0 mouse was crossed with a normal homogeneous mouse, F.
Create a mouse. For this F mouse, HL was also determined by the don't plot hybridization described above.
Hiyl the integration of the A-Bw52 gene.

From the results of don't plot hybridization confirmed in F0 and F1 mice, HLA-B is present in germ cells.
The w52 gene has been introduced and HLA-Bw is present in somatic cells.
Even if the F.52 gene has not been introduced into a chimeric individual, if the result of dot plot hybridization is positive in the F.mouse, the F.mouse is transgenic.

HLA transgenic animals are useful as experimental animals indispensable for the development of anti-HLA monoclonal antibodies and for functional analysis of HLA.

The development of anti-HLA monoclonal antibodies was based on HLA-Bw5.
2 transgenic animals were used for immunization, and the desired H
It can be carried out by known cell fusion techniques using LA antigen as an immunogen. In particular, by selecting an antigen with high genetic homology to HLA-Bw52, a monoclonal antibody with desired specificity can be developed.

For example, in conventional serological testing methods, cross-reactive groups (CREG; Cro
By selecting a combination of alloantigens belonging to ss Reactive Groups, it is possible to develop an antibody that recognizes an allospecific epitope, which has not been possible using serological methods until now.

The CREG to which Bw52 belongs is (B51. B55BW
62, B W63. Bw46. Bw53,
BW5988, B5) and the like. here,(
) stands for CREG, and the letters written therein, for example, Bw52, mean HLA-Bw52 antigen. Therefore, it is sufficient to select one antigen from CREG and use it as an immunogen.6 [Effects of the Invention] The HLA-Bw52 gene-transformed non-human mammal of the present invention can be used as an immunogen for developing anti-HLA monoclonal antibodies. They are extremely useful as animals, experimental animals, etc.

[Brief explanation of the drawing]

Figure 1 (1) and (II) are HLA-B51Bw52
FIG.

Claims (1)

[Claims] 1. HL whose germ cells and somatic cells were introduced into a non-human mammal or an ancestor of this non-human mammal at an early developmental stage
A-HLA- characterized by having the Bw52 gene
Bw52 transgenic non-human mammal.