JPH03504560A - Transgenic animals for testing multidrug resistance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Transgenic animals for testing multidrug resistance The present invention generally involves Concerning genetic manipulation. More specifically, the present invention describes the utility of expressing multidrug resistance genes. suitable for in vivo testing and development of novel chemotherapeutic agents against cancer. Concerning the production of genetic animals.

Background of the invention Intrinsic and acquired resistance to numerous chemotherapeutic agents is a major challenge in the treatment of cancer. It is a clinical problem. Numerous drugs e.g. Catharanthus alkaloids, Doxol Resistant to bicine (adriamycin), colchicine and adriamycin D Human KB Calculation After Sexual Cell Lines Are Selected in Culture for Resistance to a Single Drug A system derived from a synoma cell line (Akiyama et al., 1985. Soma t, Ce1l Mo1. Genet, 11:117-126) investigated. One human gene called MDRI for multidrug resistance is highly Multidrug resistant cell lines, elevated in some normal tissues and many tumors (Poji et al., 1987. roc, Natl, Acad.

Sci, USA 84:265-269), these tumors are located in the colon and adrenal glands. and intrinsically drug-resistant cancers of the kidney, as well as those that acquire drug resistance after chemotherapy. from a tumor. The protein product of the MD foot 1 gene is a 170 kD membrane. glycoprotein (P-glycoprotein), overexpressed in multidrug-resistant cell lines and acts as a pump to transport chemotherapeutic agents out of the cell. drug Considered as an efflux pump, P-glycoprotein is present in the cytoplasm of resistant cells. localizes in membranes, binds both cytotoxic drugs and ATP, and has been shown by sequence analysis to It has 12 domains spanning the membrane as shown in (Chen et al., 198 6. Ce1l.

47:381-389).

The full-length cloned human MDR1 or mouse mdrc DNA is of mice and humans after transfection or infection with viral vectors. It has also been reported that multidrug resistance can be imparted to drug-sensitive cells (Gros et al. , 1986. Nature 323 Near 28-731. υeda et al.

1987, Proc, Natl, Acad, Set, USA 84: 3004-3008).

However, acquired drug resistance can develop in human MD through genetic manipulation of the embryo or germplasm. Actual experimental studies have shown that somatic expression of the RI gene can occur in vivo. It had not been shown until now.

Summary of the invention Therefore, it is an object of the present invention to provide a transducer containing and expressing the human MDR1 gene. The aim is to provide genetic animals.

Another objective of the present invention is to test the effectiveness of high-grade chemotherapy against tumors. The purpose of this study is to provide an animal model for research.

Other objects and advantages of the present invention will become apparent from the detailed description hereinafter. There will be.

Brief description of the drawing The above objects and other objects and aspects and many advantages of the present invention are as follows: BRIEF DESCRIPTION OF THE DRAWINGS It will be better understood from the following detailed description taken in conjunction with the drawings.

Figures IA and IB are schematic diagrams of the construction of expression vectors pHG1 and pHG2. Ru.

Figure IA shows the construction of pH01 and isolation of pronuclear injection fragments.

Figure IB shows the construction of pH02. Characters are used for cloning and orientation characterization. Restriction enzymes used for this purpose are shown: B, BamHI; E, EcoRI ;N, Ndel;S, 5ail;X.

Show rank.

Figures 2 and 2B show Southern hybridization of tail genomic DNA from mice. The results of the analysis are shown below.

Figure 2A shows the B-actin promoter MDR1 fusion integrated into the mouse genome. It is a schematic diagram of a transgene. If the transgene is integrated , EcoRI digestion of mouse genomic DNA and extraction from the central region of MDR1 cDNA. 3. After hybridization with the derived 5A probe. lkb label cleavage Fragments are expected.

Restriction site symbols and letters are the same as those described in FIG.

Figure 2B shows digestion with EcoRI and low stringency with 5A probe (rows 1- 5) and after hybridization under high stringency conditions (rows 6-10). Southern blot analysis of genomic DNA (20 μg) is shown. Each genomic DNA Isolated from: KB-1-1 cells (rows 1, 6); injected DNA fragment 1 Normal mice mixed with 09g (rows 2, 7); normal mice (rows 3, 8); Negative mice from a litter born after injection (row 4°9); genic mouse (row 5°10).

Figures 3A and 3B show DNA from founder mice carrying the MDRI) lance gene. FIG.

Figure 8A shows hybridization under denaturing and high stringency conditions with the 5A probe. DNA slot plot analysis of 0 μg of tail DNA after injection is shown. NM, normal ma Us, MB2-M2B5. ) Transgenic founder mouse; IC or 10C1 Transgene copy number as described for columns 2 and 7 in Figure 2B. show.

Figure 3B is a Southern blot analysis of tail DNA from the founder mouse described in Figure 3A. shows. No. 1 hybridization was performed under high stringency conditions with the 5A probe. Ta. The stamp is 8. lkb fragment; arrow indicates fragment of transgene rearrangement product. Show the piece.

Figures 4A and 4B show MDRI) runs in mice born from founder M39 (Figure 3). Indicates the inheritance of the bus gene.

Figure 4A shows the pedigree of MB2 and descendants. Square, male; round, female; half black symbol, carrier mouse: white blank symbol, non-carrier.

Figure 48 shows Southern blot analysis of tail DNA from F2 generation mice. mouse The numbers correspond to the numbers shown in the genealogy above. Molecular weight standard (lkb ladder) (B RL) is shown.

Figure 4C shows tail DNA from mouse 39 and its Fl and F2 generation progeny. Slot plot analysis is shown.

Probes and hybridization conditions were as described in Figures 3A and 3B. is the same as

Figure 5 shows strain expression analysis of the MDR-39 mouse line.

Square, male; circle, female; white blank symbol, non-carrier mouse; bottom half marked with black blank. No., heterozygote; symbol entirely black, homozygote; symbol with diagonal line in the upper half, R Mice expressing the transgene as detected by NA test; upper half of the mouse symbol, mouse expressing the transgene upon detection by protein immunofluorescence localization. vinegar. Ne, mice that express the transgene in the lungs but not in the bone marrow. letter The mice marked with are those for which RNA testing is shown in FIG.

Figure 6 shows the results of RNA expression analysis of normal mice and transgenic mice. vinegar. Extracted from bone marrow and lungs of mice A-E (as shown in Figure 5). The slot plot of the total RNA sample (10 μg) is shown with the MD lobe (right side). Hybridized under high and low stringency conditions, respectively. 3- 1. Parental drug-sensitive KB cell lines; 8-5 and v-1, multidrug-resistant supply lines; BM, bone marrow; SP, lung.

Figure 7 shows tissue specificity of transgene expression.

Figure 7A shows a schematic restriction map of the MDR1 cDNA and probe used. E, BcoRI; P, PVUII.

Figure 7B is a plot of the total RNA sample (10 μg) extracted from Showing hybridization: Upper side: 1. KB-8-5 drug resistant cell line, 2. KB-3-1 drug-sensitive cell line and KB-V-1 drug-resistant cell line; Bottom: 1 ．． MDRI non-carrier mouse; 21MDRI carrier mouse. Li, liver H Ki, kidney; LU, lung; Ov, ovary; Mu, skeletal muscle; BM, bone marrow; sp.

Internal organs; He, heart HBr, brain. The same plot with different probes as shown hybridized.

Figures 8A, 8B, 8C and 8D are MDRI) bone marrow from transgenic mice. Immunofluorescence localization of human P170 in cells is shown. MDRI) Lancegenic Mau either from a littermate (Figures 8A, 8B and 8C) or from a normal litter (Figure 8D). Bone marrow samples were spread on glass slides, air-dried, and fixed with formaldehyde. Set.

The applied material was labeled using monoclonal antibody MRK16 (anti-human P170), and the and indirectly label with rhodamine. from MDR mice by exposure for the same time. Bright expression of P170 is seen in all cells (Figures 8A', 8B' and 8C'), absent in cells from control mice (Fig. 8D'). (Figures 8A, 8B , 8C and 8D) are shown in (Fig. 8A', 8B', 8C' and Fig. 8D°). It represents a phase-contrast image of a cell. (Magnification = xsso, bar = 6.5m) Detailed description of the invention The above object and various other objects and effects of the present invention are directed to the human MDRI gene. This is accomplished by transgenic animals that have offspring and express.

Unless otherwise specified, all technical and scientific terms used herein refer to the present invention. Similar to what is normally understood by a person of ordinary skill in the art (person skilled in the art) have meaning. Any similar or equivalent methods described herein and Although materials can be used in the practice or testing of the invention, preferred methods and and materials are described here. All publications mentioned below are incorporated by reference. It will be incorporated into the specifications. Unless otherwise specified, techniques used herein are within the skill of those skilled in the art. It is a well known standard methodology.

This was the first step toward creating transgenic animals.

Both humans and mice have one functional actin gene per haploid genome. Actin exists in eukaryotic cells, but it is related to the origin of the embryo. One of the most abundant proteins and the cutin promoter in various tissues requires both of the following: It was considered that the levels of expression and activity could be met. The following materials and methods Generation and testing of MDR1-expressing transgenic mice according to the present invention 1 illustrates various stages in the process.

material and method material Restriction endonucleases and T, DNA ligases were prepared by two English manufacturers. purchased from Iolapus or Bethesda Research Laboratories, Inc., and Used under conditions recommended by supplier. Agarose (Sechem, GTG) and Sea Plaque, LMB) are supplied by FMC (Rockland, Maine). It was. Reagents for PAGE were from Piorad. colchicine is sigma -Purchased from Chemicals. All other chemicals were of analytical standard. . Cell culture medium was obtained from Gibco, and serum was obtained from Em, Nee, BioProducts. Obtained from TS. (P”)-dcrp and nick translation kit was purchased from Amersham or Niney England Nuclear.

Bacterial strains, plasmids, cell lines and mouse strains E. coli strain D85 for transformation used. The full-length (7)MDRIC DNA was combined with a single-Xho1 site at the 8' end. pMDR2000XS was constructed in our laboratory (Pastan et al. 1988. Proc, Natl, Acad, Set, USA 85:4486-4490). The pGEM2 vector was produced by Promega Biotik (La. (Madison, Isconsin). Chicken-actin in pUc18 pBA-CAT, which grows the bacterial CAT gene under the control of a promoter, was developed by B. Pat. A gift from Dr. Son. 10 NIH8T3 and KB-3-1 cells each Growth in Dulbecco's modified Eagle's medium supplemented with % fetal bovine serum and bovine serum. I let it happen.

Mouse strains RCNIH and B6SJL F were manufactured by Jackson Laboratories (Med. Obtained from Per Harbor, Hone.

Plasmid construction Built to be. Chicken Dan-pBA with actin promoter- CAT is cleaved at 5all and contains the essential actin promoter sequence. A 0.33 kb fragment was eluted after electrophoresis on an agarose gel. pGEM 2 vector (Pa5tan et al., 198B, Proc, Natl.

Acad, Sei, IJsA 85:4486-4490) A single cell of pMDR2oooxs containing the full-length MDR1 cDNA downstream from the %-tar. The generated fragment was inserted into the -5a11 site. was used to identify the produced mido.

The resulting plasmid was used for p-injection. pHG-1 is micro Before injection, the actin is digested with Xhol (see below), and actin is digested with Xhol (see below). The effect of truncating the promoter was determined. Therefore, once the tip is cut, the actuator To test the expression level of MDRI under the control of the pHG- 2 (AP-MDR) was constructed from pHG-1 as shown in Figure IB.

pHG-1 was cleaved with Xhol, and the resulting 4, 8 kb and 2.9 kb fragments were assembled. It was eluted after separation on a galose gel. 6 of the 5' region of the B-actin promoter Cut the 2.9 kb fragment containing 0 base pairs with 5ail and remove these 60 bp sequences. Xhol-Sail (27 0bp) 4.8kb containing MDRlcDNA downstream from the truncated b%-9- The fragments were concatenated. The resulting plasmid contains multiple clones of pGEM2 vector sequences. The actin promoter-MDR1 sequence is located at the 5′ end of the binding site in two orientations. It had

Restriction analysis with Xhol/Ndel was used to isolate T7 of the pGEM2 vector. It was done. The generated plasmids pHG-2 (tBAP-MDR) and pHG-1 were Their size and human KB-3-1 and mouse NIH8T3 transfectants The expression of MDR1 cDNA in the cells was tested.

Cell culture and transfection NIH3T3 mouse cells and human KB-3-1 cells were cultured and 5hen et al., 1986. Mo1. Ce1l Biol, 6:4039-404 Transfection was performed by the calcium phosphate precipitation method described in 4.

DNA preparation for microinjection: Cleave pHG-1 with Xhol, and First, a 4.7 kb fragment containing MDRI downstream from the actin promoter was isolated. Vector cleavage was performed by electrophoresis on a spot agarose gel (see [Plaque J below)]. separated from the pieces. The ethidium bromide stained band containing BAP-MDR-1 was removed from the gel. and TE (10mM) Lis-HCl pH 8,0,1mM EDTA) in equilibrated phenol solution at 70°C. Then 0.25M Add 3 volumes of buffer containing NaCl, TE and incubate at 70°C for 10 min and 40 min. Incubate at ℃ for 10 minutes. Separate the aqueous phase and extract into chloroform (1:1 v/v) and ethanol precipitated. DNA pellet 0.2M NaCl, T and on a NACS column (Bethesda, Research Laps). Supplied except that DNA was eluted with 1.0M NaCl, TE, and 1% caffeine. Purified under conditions recommended by the author. Ethanol precipitate the eluate and remove excess salt. Removed, washed with 75% ethanol and 95% ethanol, and dried D NA pellets were dissolved in sterile HtO. Mix the DNA sample against sterile H,O. Microdialysis was performed for 30 minutes using a Lipore filter (#VMWP 01300). sample was removed from the filter, centrifuged for 15 minutes in an Eppendorf centrifuge, and 2/3 volume was collected from the upper layer. The concentration of the DNA sample was determined by gel electrophoresis. Determined using Thidium-stained DNA standard (λDNA/Hindlll fragment) . Finally, 7゜3mM PIPBS pH7,4,0,1mM EDTA , dilute the DNA with microinjection buffer containing 5mM NaCl. I interpreted it.

Microinjection of fertilized eggs into mouse fertilized eggs using B6SJL F. 1-2 ng of DNA was removed from the oviduct of a female and microinjected. I turned on. The microinjected embryos were transferred to CRNIH surrogate females.

Mouse and egg manipulation and microinjection techniques were described by Hogan et al. 1986. "R Mouse Embryo Manipulation: Experimental Manual" by Cole, New York Cold Spring Harbor, Cold Spring Harbor Laboratory) It was done as if it were.

Preparation and analysis of mouse genomic DNA High molecular weight genomic DNA was isolated from 1-2 inch tail samples by standard methods. Eco 20 μg of the DNA sample digested with R[ electrophoresis on nitrocellulose paper and gel electrophoresis on nitrocellulose paper. Hern, 1975. J, Mo1. Biol, 98:503-517) It was moved by RNA 5hen et al., 1986.5science 232: 1% agarose/6% formaldehyde gel as described in 643-645. Electrophoresis was carried out inside. 0 μg of total RNA was injected per row. Liposome R Only samples in which the NA appeared intact were analyzed. Maniatis et al., 198 2. "Molecular Cloning: Experimental Manual" Coldspring, New York Cold Spring Harbor, Cold Spring Harbor Laboratory) Transfer RNA to nitrocellulose paper or nitrous membrane (Schleicher and Schnill Inc.). For semi-quantitative analysis, RNA samples were analyzed by Fojo et al. , 1987. Proc, Natl, Acad, Sci, USA 84:265-269 using a slot plot device. applied to the filter by

Hybridization is as described for DNA hybridization. It was conducted. Northern and slot plot filters 0.2XSSC, 0, low shrinkage at 50°C in 1% SDS) or 70°C in O,1X5SC,0,1% SDS (high austerity) and washed as indicated in the text.

are obtained from contact areas and account for approximately 85% of all messages. Schematic cDNA The map is shown in Figure 7A.

Probe 10.1.6 kb fragment was obtained by EcoRI digestion of pMDRIO. Nibrope 5A, a 1.4 kb fragment obtained by EcoRI digestion of pMDR5A. and probe PVU2#6 was pMDR2000 (Ueda et al., 1987 ゜Proc, Natl, Acad, Sci, USA 84:3004 -3008) obtained by purifying the 0.84 kb fragment from the PVUII digestion of It was done.

Immunofluorescence localization Bone marrow cells were plated onto glass slides, air-dried, and then incubated in 3.7 ml of phosphate buffer. % formaldehyde for 5 minutes at 23°C. Control or transgenic mama Slides from mice were then incubated with mouse monoclonal antibody MRK16 and then was incubated with rhodamine-conjugated affinity-purified goat anti-mouse IgG (Wi 11 Ingham et al., 1987, J. Histochem, Cyto chem, 35:1451-1456).

result was inserted into the 5′ side of the MDR1 cDNA (Fig. IA), and the raw rotor ss ob p followed by -140 (Sac1 site) to +4240 (BcoR1 site) Contains MDR1 cDNA up to 438 obp. Cut the 4690 fragment with Xho I. It was cut into pieces and used for microinjection tests.

This fragment contains multiple clones from pGEM2 at the junction of the actin promoter. The sObp at the 5° end of the 24bp region of the rowing site is microinjected. not present in the edited fragment. dan-remaining 270b of actin promoter Although p contains a consensus CAAT and TATA box, this truncated It was not known whether the fragment expressed MDRl. Therefore, this tip Contains MDR1 cDNA downstream from the truncated promoter but is otherwise identical. An expression vector (1-BAP-MDR) was constructed to test the expression of MDRI. It was done.

Transfection test under the control of the B-actin promoter, as well as the truncated B-actin promoter. To test the expression level of MDRI under the action of 1 and pH02 were stably transfected into drug-sensitive KB-3-1 cells. (Table 1). p　Ha　M　DR　R.

A retrovirus expression vector containing two Ha-MSV LTRs was used as a positive control. and used it. For negative controls, cells were similarly transfected in the absence of DNA. The treatment was performed according to the following protocol. A similar negative result was obtained using a vector without the MDRI sequence. also obtained after transfection using target DNA (data not shown). do not have).

A dish of KB-3-1 cells (IXIO' cells/10 an dish) was treated with calcium phosphate. Using 10 μg of either pHG1, pH02 or pHaMDR, use the precipitation method. transfection and varying concentrations of colchicine (parental KB-3-1 cells). LDs of colchicine (55-8 n/ml), 3-5 times higher than selected. The tests summarized in Table 1 were resistant colonies to 51 g/m 1 colchicine. shows comparable results for AP-MDR and pHaMDR once p in the number of . However, the average colony size was The pH value for the transfection form was smaller than that for the transfection form (pHaMDR). Ta. This difference in size as well as the difference in colony number at high concentrations of colchicine is due to induced in KB cells. However, once the tip is truncated - the actin promoter ( Expression of MDR1 under the control of the full-length AP-actin promoter (AP-MDR) -comparable to or higher than the expression of MDRl under the control of -.

Confirm that the Dan-actin-MDRI expression vector functions in mouse cells For this purpose, these vectors were introduced into NIH3T3 cells and the parental NIH3 60 ng/ml colchicine in T3 cells and transfectants (relative to parent cells) LD of colchicine. Colony forming ability was measured at 3-5 times higher than that of this In a study (data not shown), a similar relative in the presence of colchicine Colony-forming ability of NIH3 with full-length or truncated actin promoter constructs Tl) obtained for transfectants, therefore, the truncated end-actin The promoter drives the expression of MDR1 cDNA in transgenic mice. It was concluded that it is a suitable promoter for

Microinject the fragment (Figure IA) into B6SJL (Fl) fertilized eggs. MDRI) transgenic mice were born. Tail genome digested with EcoRI Mice were screened by Southern blot analysis of muDNA.

The plots were hybridized with the 5A probe (Figure 2A). Figure 2B is Southern blot analysis of tail DNA from several mice is shown, indicating whether MDRlcDNA or as well as the predicted single 3.1 kb internal fragment of Mouse endogenous fragments detected under high stringency conditions (columns 6-10) It was seen. For each plot analysis, dilute the full-length 4.7 kb injected fragment and mixed with mouse genomic DNA, digested with EcoRI, and completely isolated the genomic DNA. [! Injected into the gel as an internal control for coRI digestion. This sample also has large Indicates the size and MDRI copy number in each transgenic mouse. used as a standard for estimation (columns 2, 7). Human KB-3-1 genome D EcoRI digestion of NA was also used to confirm the estimated MDRI copy number. I was able to do it (columns 1, 6). DN human samples from MDRI-positive mice were also slotted. MDRI in each transgenic mouse was analyzed by plot analysis. Confirmed copy number.

In this study, five founder mice containing integrated human MDRI sequences were born. (Figure 3): Mouse 39 (female) has a low copy number (1-3 copies). Mouse 132 (female) contained 100-200 copies of MDRI; Mouse 168 (male) had approximately 50 copies of integrated DNA; 93 (male, high copy number) showed additional MDRI fragments in Southern analysis. After weaning It died soon; mouse 104 (female, about 10 copies) was the first of its own offspring. She killed her child and died during her second pregnancy.

Transmission of MDRI to progeny The remaining three transgenic founder mice (Mg2, M2B5 and M1 6B) with normal mice and the tail genome D from their progeny. NA samples were analyzed by Southern blot hybridization for the presence of MDR1 sequences. and analyzed by slot plot hybridization. two animals The original mice (Mg2 and M2B5), as expected, had integrated MDRI sequences. was transmitted through the germ line to approximately 50% of the progeny. number of DNA sequences obtained or No significant changes in structure were detected in the Fl and F1 generations (Fig. 4; data not shown). (Not done). It was shown that the third origin (M168) was present. Therefore, It was concluded that this third founder mouse was probably a mosaic.

MDRI) Expression test in transgenic mice Founder mouse M39 (low copy ) and its positive progeny were crossed with normal mice and named MDR-39. A MDRl heterozygous mouse line was produced. This system was selected for RNA expression studies. It was done. The total RNA was 7 F.

Transgenic mice (see pedigree in Figure 5) and 10 negative littermates 18 different tissues (brain, liver, kidneys, lungs, heart, lungs, stomach, small intestine, colon, skin) prepared from skin, tail, bones, bone marrow, skeletal muscle, ovaries, uterus, fallopian tubes, and testes).

These RNA samples were subjected to slot plot hybridization with the MDR1 probe. (Figures 6 and 7). Accurate RNA injection for all plots and cross-linked with B-actin probe as a standard control for filter transfer. hybridization (Figure 6; data not shown). If a known sample is known MDRI RNA content and can be directly compared with known multidrug resistance samples. Multidrug resistant individuals expressing elevated levels of MDRl mRNA in each experiment Total RNA from a sexual KB cell line was included. Compared to drug resistant KB-3-1 Therefore, the multidrug-resistant supply line KB-8-5 has a 40-fold increase in MDRl mRNA. and KB-V-1 (Vb1) has an increase of more than 500 times. ing. These experiments, summarized in Figure 6, show that humans. low expression was also detected in skeletal muscle and ovary (Fig. 7B), but not in testis. (data not shown). The same plot, together with MDRlcDN Three different contiguous MDR1 probes (10,5A and PVU 11 #6). 5' probe (pro Scaffolds that hybridized more markedly to 10) than to others With the exception of muscle, all positive tissues showed similar results with all three probes (Figure 7B). These results apply to all groups except skeletal muscle expressing human MDRI RNA. Figure 2 shows that the entire MDRl mRNA was expressed in the tissue. northern plot analysis (data not shown) of 4.5 kb (full-length expressed MDRI protein). showed an indeterminate message size ranging from approximately 1 lkb. these The results suggest that the endogenous polyadenylation signal at the 3″ end of the MDR1 cDNA This shows that it had a weak effect on transgenic mice.

Of the seven MDR-39 mice tested, six showed significant expression in the bone marrow. and showed somewhat lower expression in the lung. However, one mouse It showed expression in lung but not bone marrow.

One male mouse showed high signal in skeletal muscle and bone marrow and lungs. In addition, it showed some expression in kidney and liver. Some expression is trans Detected in the ovaries of genetic females, but not in the uterus or oviduct. , no testicular expression was detected.

Expression of P-glycoprotein detected by immunofluorescence was induced from control littermate mice. mouse bone marrow for human P-glycoprotein (MRK-16). Immunofluorescence with monoclonal antibodies showed that two MDRI-positive mice had many different Figure 8 shows the surface expression of human P-glycoprotein in bone marrow cells. This thing is , background detected in application using rhodamine-conjugated anti-mouse IgG. Despite the presence of 1gG in mouse plasma, it cannot be clearly seen in Figure 8. came.

In summary, the results presented here demonstrate that human MDR1 under the control of a heterologous promoter This clearly demonstrates the success of creating transgenic mice carrying and expressing cDNA. . The promoter itself appears to be a structural promoter, and the 3′ of the promoter region and located 5′ of the polyadenylation signal of the B-actin gene itself. Only descending control is performed in a small area. Any of these inhibition regions may be affected by the structure of the present invention. It was not included in the structure. Cloned bacteria chloramphenicol acetyltran Induction of expression vectors containing the spherase (CAT) gene into a wide range of cell types. Comparative studies of DNA-mediated transport show that once-actin promoter activity lomotor (Gunning et al.

1987, Proc, Natl, Acad, Set, USA 84: 4831-4835). Dan - actin is cultured Since it is abundantly expressed in all cells in the nutrient solution, once the control of the actin promoter is activated, Transgenes containing MDRI sequences exhibit high levels in a wide range of cell types. It was thought that the drug may be expressed in many cases, and therefore may confer drug resistance. Try this prediction To test, first, the Dan-actin MDRI expression vector (pH01) was injected into cell culture. The ability to confer drug resistance phenotypes in nutrient solutions has been tested, and indeed significant acquired Multidrug resistance was observed.

The presence of prokaryotic vector sequences indicates that the presence of certain genes in transgenic mice It was shown to be highly inhibitory to proper expression. In addition, prokaryotic vectors In mice carrying the transgene adjacent to the target sequence, chromosomal abnormalities e.g. Localization instability at the site of integration and insertional mutagens is observed. To the present invention To excise vector sequences in such pronuclear injected DNA, Xho of pHG1 The l fragment was isolated. This strategy also targets the 5′ region of the B-actin promoter. 6 obp was excised.

A plasmid (pH02) lacking the sObp upstream of the target was constructed and injected into the culture medium. were transfected into human and mouse cells. Through these studies, The activity of the truncated B-actin promoter is similar to that of the full-length promoter in cultured cells. etc. or higher. Therefore, it was found that.

One line (MDR-39 ) were selected for expression studies. This founder and over 100 descendant mice? Restriction fragment pattern analysis of their genomic DNA (Fig. 5; data not shown) , indicating that 1-3 copies of the injected fragment were integrated into mouse DNA. Also, No internal rearrangements, deletions or duplications were detected. Transgenetics through genealogy analysis Offspring = (a) transmitted by the germ line; e) transgenes are heterozygous and A single stain is stably inherited by approximately 50% of the progeny from mouse mating. integrated into the color body; and (C) transfer of the transgene from male to male. and female-to-female transmission occurs; therefore, it is inherited autosomally, and It was shown that there were no chains.

Insertion of foreign DNA sequences into a cell's genome creates an endogenous gene or control element. It is known that mutational changes can be induced by disrupting the function of a protein. Tiger Most insertional mutations in genetic mice are recessive, but heterozygous In most crosses between zygotic mice, their embryonic lethal phenotype is significantly reduced. indicated by low litter size and lack of ability to produce homozygous mice. . Homozygous matings of MDR-39 progeny showed normal litter size and expressed in this mouse strain, since homozygotes for the type are usually born (data not shown). Insertion mutations in endogenous housekeeping genes or control genes are transgenic. It was concluded that this does not occur during integration of the bus gene.

RNA testing of MDR-39h Lanzienik mice revealed that the transgene was hematopoietic. It has been shown to be primarily expressed in tissues such as bone marrow and pancreas. Low expression is Detected in skeletal muscle and ovaries. However, phenotypic changes and morphology Transgenic mice examined for functional changes as well as functional or behavioral perturbations was not detected.

Human-specific 35 base synthetic oligonucleotide (mouse mdr sequence under high stringency conditions) (Does not cross-hybridize to) (Ueda et al., 1987. J. Bjol, Chem, 262:505-508) A single approximately 192 base stretch, as expected from detection from tissues expressing occured. These results demonstrate that mouse endogenous promoter regulation adjacent to the integration site A single major transducer under the control of the actin promoter Indicates the transcription start site of the gene.

MDR-39) Quantitative measurement of RNA expression in the bone marrow of transgenic mice It showed relatively high expression of MDRI. Expression level in KB-8-5 drug resistant cells expression levels of MDRl mRNA in some mice or It is up to three times higher in Japan. These levels (basal expression in drug-sensitive cells) 40- to 120-fold higher than 6MDR levels) in multidrug-resistant human tumors. at least as high as IRNA levels.

Combined, the present invention provides chemotherapy that is toxic to the bone marrow by expressing the human MDRI gene in bone marrow cells. to be tolerant to the administration of the method. For example, the chemotherapeutic drug daunomycin Intraperitoneal administration resulted in bone marrow injection 2-3 weeks after treatment in the majority of mice so treated. leading to oppression and death. In contrast, the transgenic mouse of the present invention has normal Resistant to administration of chemotherapeutic agents that are lethal to mice. Therefore, MDRI By establishing tumors in transgenic mice (transplantation or by introducing genetic changes that make mice more susceptible to forming such tumors, or by other appropriate treatments. (by appropriate methodologies) of preparations that may contain a single drug or a combination of drugs. A range of acceptability may be determined.

The transgenic mice of the present invention can also be used to carry MDR-39 or MDR-39 for various purposes. and other genetics that improve the applicability of MDRI transgenic mice. Enables child sweeping. These manipulations produce mice homozygous for the MDRI locus. Generate sufficient close-circuit strains of mice for tumor xenograft studies and for tumor xenograft studies This includes the breeding of MDRI mice to Additionally, MDR1 mice were exposed to nude immunity. By breeding with deficient mice, they are still multidrug resistant and do not accept human tumors. You can get animals that are

This allows testing of the tolerability and efficacy of anti-tumor or anti-cancer drugs. Ru.

Other uses for cloned MDRIs include human and animal bone marrow and other tissues. as a potent selectable marker to introduce linked genes into the protein. MDR1 Mau agents required to spread the disease and introduced into the recipient host (e.g., daunomycin). Shin, vincristine, vinblastine, VP-16, VM-26, Adriama Ishin D.

adriamycin, etc.). This is because the MDR1 gene of these and other chemotherapeutic agents on the bone marrow and other tissues that express This is done by using MDR1 mice to determine tolerated doses. rice cake Of course, such studies would be useful to use the MDRI gene as a selectable marker. is needed before the development of a complete gene therapy regimen.

MDRI) transgenic mice also contain the polynucleotide encoded by this gene. Enables the study of the role of drug transporters. in tissues where this gene is not normally expressed. Expression of the MDRI gene at relatively high levels in the Allows determination of physiological effects.

Furthermore, transgenic animals (MDRI) that express the human MDR1 gene can act as a donor for cells and tissues. These cells are established in culture or be used as a source of multidrug-resistant tissue in animal transplantation experiments. .

The examples and implementations described herein are for illustrative purposes only; Various modifications and changes can be made by those skilled in the art based on the description in this specification. are included within the spirit and scope of this application and the appended claims. It is understood that this is something that should be done.

Table 1 Frequency of drug-resistant colonies Mouth==ko MDRl Mouth　　　　　　　B 7rittD me? -5 脣ts 7D CAT 1 6’L ■ T7 Aξ-7-RO SP6γD Me? − 8, 1 2345 6 78910 Amendment translation submission form (Patent Law No. Article 184-8) April 22, 1991 1. Display of patent application PCT/US 89104686 Z invention name Transgenic Animals for Testing Multidrug Resistance 3, Patent Applicant Name United States of America 4. Agent Address: Ochanomizu Square Building B, 1-6 Kanda Surugadai, Chiyoda-ku, Tokyo Name (6861) Keio Kei (and 1 other person) Scope of claim 1. Transgenic mammalian reproductive cells and body tissue.

2. Transcription of the human 1 gene is under the control of the truncated dan-actin promoter The non-human transgenic mammal according to claim 1.

3. When the tip is truncated - actin promoter is full length - 5° of actin promoter 3. The non-human transgenic of claim 2, which is devoid of about eot = shadow from the region. mammal animal.

4. The non-human transgenic mammal according to claim 1, wherein the mammal is a dentodont. mammalian animal.

5. The non-human transgenic mammal according to claim 2, wherein the W toothed cervix is a mouse. animal.

6. (a) Transferring a tumor to the non-human transgenic mammal according to claim 1. plant or introduce; ('b) in the plurality of groups of mammals of step (a), the amount increased in each group of mammals; administering chemotherapeutic agents alone or in combination, (C) Administration of chemotherapeutic agents that prevent tumor growth or proliferation without intolerable side effects. determining the dosage and length of testing high-dose chemotherapy for tumors consisting of determining the maximum tolerated dose of the chemotherapeutic agent; and (b) After the multidrug-resistant somatic cells are introduced into the host mammal, there are Using the maximum tolerated dose of multidrug-resistant somatic cells containing the DRI gene, Select by Gene transfer in which the MDR1 gene is used as a selectable marker How to test protocol effectiveness.

8. Ta) A chemotherapeutic agent in which MDRI-expressing cells grow in the presence of the chemotherapeutic agent Determine the maximum tolerated dose of (+4(b) Multi-drug resistant somatic cells in mammals can be detected using the maximum tolerated dose. choose from, Methods for testing the effectiveness of gene transfer protocols used in

9. Human rat pellet obtained from the non-human transgenic mammal according to claim 1 A cell or tissue that expresses a single gene.

international search report PCT/! ;Sei'+/u46ac+trar+sgenic, actin , MDtll+beta, chiegan, anizal, 5ere enir+g.

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Claims (5)

[Claims] 1. A transgenic animal that has and expresses the human MDR1 gene. 2. The animal according to claim 1, which is a mouse. 3. (a) transplanting or introducing a tumor into the transgenic animal according to claim 1; (b) administering a chemotherapeutic agent to the plurality of groups of animals of step (8) in increasing amounts to each group of animals; alone or in combination; (c) tumor growth or Determine the dose of chemotherapeutic agent to prevent growth, consisting of a high dose to the tumor How to test chemotherapy. 4. (a) MDR1-expressing cells grow in the presence of the chemotherapeutic agent; determining the maximum tolerated dose; (b) multidrug resistant somatic cells are introduced into the host animal; Later, the multi-drug resistant somatic cells into which the MDR1 gene has been incorporated are subjected to the maximum tolerance Select by using volumetric dosage, Gene transfer in which the MDR1 gene consisting of How to test protocol effectiveness. 5. A cell expressing the human MDR1 gene obtained from the animal according to claim 1, or organization.