WO1997018703A1 - Method for constructing transgenic animals - Google Patents

Abstract

A method for conveniently and efficiently constructing transgenic animals comprising infecting a fertilized animal egg for which the egg membrane has been eliminated with a non productive recombinant Adenovirus having a foreign gene introduced thereinto and then transplanting the embryo into the uterus of a host.

Description

 Description How to make transgenic animals

Technical field

 The present invention relates to a transgenic animal obtained by introducing a foreign gene into a genome using a non-replicating recombinant adenovirus, and a method for producing the transgenic animal.

Background art

 Creation of transgenic animals has been more difficult than before, and at present the facilities that can produce the intended transgenic animals are extremely limited.

 Therefore, development of a simple and efficient method for producing transgenic animals is strongly desired.

 On the other hand, the present inventors have found that non-propagating (E1A deficient) recombinant adenovirus is hardly integrated into the host genome (Rosenfeld et al., Science—252, 43 1-434 (1991)). Various studies focusing on the fact that it has been used for gene regulation research and gene expression for the development of somatic cell gene therapy (Akli et al., Nat. Genet. _3, 224-228 (1993)). In addition, it was confirmed that a non-replicating adenovirus containing the LacZ gene could infect pronuclear-stage non-enveloping mouse eggs and was expressed without any adverse effects in the cleavage-stage preimplantation embryo. (Tsukui et al., J. Ma Rat Ova. Res., 10 (1), (1993, 4) 150-151).

 However, expression was transient even by this method, and stable transgenic animals were not successfully produced (Tsukui et al., Ol. Reprod.

Dev. 42, 291-297 (1995)).

 Therefore, a first object of the present invention is to provide a simple and efficient method for producing a transgenic animal in which a foreign gene has been introduced into the genome. Further, a second object of the present invention is to provide a transgenic animal produced by the method of the present invention.

Disclosure of the invention The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a non-replicating recombinant adenovirus was integrated into the genome of an egg of an animal, and the integrated adenovirus DNA was incorporated into an animal germ cell. It was discovered that transgenic animals could be produced by transferring to a system, that is, by using a non-replicating recombinant adenovirus. The present invention has been completed based on such findings and further studies.

 That is, the gist of the present invention is:

 (1) A transgenic animal characterized in that a non-replicating recombinant adenovirus carrying a foreign gene is infected into a fertilized egg from which the egg membrane has been removed, and then the embryo is transplanted into the uterus of a parent animal. How to create

 (2) The transgene according to the above (1), wherein the foreign gene is a nucleotide having a base sequence encoding a chromogenic protein as a bioactive protein, an antibody, or a marker gene or a drug-resistant protein as a marker gene. How to make animals,

(3) above, wherein the dosage in the case of a non-proliferating recombinant adenovirus Ru were infected with an animal embryo removal of the egg membrane is ^{5 X 1 0 7 ~1 0 8} pf / 1 (1 ) Or the method of (2) for preparing a transgenic animal,

 (4) The method for producing a transgenic animal according to any one of (1) to (3), wherein the non-replicating recombinant adenovirus is expressed under the control of a CAG promoter.

 (5) The method for producing a transgenic animal according to any one of (1) to (4) above, wherein the non-proliferating recombinant adenovirus contains a nuclear transport signal.

 (6) The transgene according to any one of (1) to (5), wherein only one copy of the non-propagating recombinant adenovirus into which the foreign gene has been introduced is integrated into the genome of the animal fertilized egg. How to make animals, and

 (7) A transgene created by infecting a fertilized egg with a non-proliferating recombinant adenovirus into which an exogenous gene has been introduced into an egg-removed animal and transplanting the embryo into the uterus of a borrowing animal Animals,

About. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a photograph of an electrophoresis showing the identification of integration of a non-replicating adenovirus DNA by Southern analysis. Ec Genomic DNA isolated from the tails of 27 animals. The digest was digested with a shakupobi ^! IndIII, and the cosmid DNA of pAxCANLacZ, which has 98% homology with the AxCANLacZ sequence, was used as a probe. a is EcoRV digestion data, b is Hind1 [1 digestion data. Open circles are end fragments of adenovirus. The asterisk indicates the connected fragment of the virus-cell DNA after the integration event.

 FIG. 2 shows adenovirus DNA integrated into the genome of three transgenic mice. The thick line shows the restriction enzyme map of the recombinant adenovirus AxCANLacZ.

 FIG. 3 is a photograph of electrophoresis showing germline inheritance in F1 progeny and expression of the adenovirus transgene on F1. LacZ expressing transgenic mice (AC L9.23) were bred to ICR females and the 10 F1 fetuses that developed were tested 13.5 days after gestation. Genomic DNA isolated from the 10 placentas was subjected to Southern analysis according to a procedure similar to that shown in FIG. Lanes 1, 3, 4, 7, 8, and 10 indicate that there was transmission of the adenovirus transgene, and lanes 2, 5, 6, and 9 indicate that there was no such transmission. The asterisk indicates the ligated fragment of virus-one cell DNA after the integration event.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail.

 ^ The proliferative adenovirus used in the present invention was Hiromi Kanegae, Shizuko Harada and Izumi Saito "Experimental Medicine Supplement" Bio Manual Series 4 "Gene Transfer and Expression ♦ Analysis Method" 4 page 3-58 "Adenovirus was used. Gene Transfer ”, 1989. A non-propagating adenovirus into which a foreign gene has been introduced can be prepared.

The non-competent adenovirus used in the present invention has several features. In other words, adenoviruses have a very wide host range, and secondly, allow the introduction of recombinant genes into non-dividing cells. The non-replicating recombinant adenovirus has such properties and is expected to be a vector for fertilized animal eggs in the pronuclear phase. In our study, we used Adex4SRLacZL containing the E. coli LacZ gene under the control of SRa Promote, a non-reproductive adenovirus vector. Attempts to introduce a gene into the fertilized egg at the pronucleus stage failed to detect expression in 13.5-day embryos that grew from infected eggs (Tsukui, T., et al. Ol.

Reprod. Dev. 42, 291-297 (1995)).

 In the present invention, it is necessary to introduce a promoter into an adenovirus vector in order to enable expression of a foreign gene after infecting animal cells. Any of those that have been used for gene transfer using E. coli can be used, and in particular, CAG promoter (Araki et al., Proc.

Natl. Acad. Sci. U.S.A. 92, 160-164 (1995)) is preferably used.

 In the present invention, if toxicity due to accumulation of the expression product of the foreign gene in the cytoplasm is concerned, a nuclear localization signal sequence may be incorporated together with the foreign gene. The nuclear localization signal sequence is expressed by the adenovirus vector in the nucleus of infected cells and promotes the translocation of foreign gene products secreted outside the nucleus into the nucleus again (Daniel Kalderon et al., Cell 39, 499- 509 (1984)).

 In order to clarify the result of virus integration, the present inventors use AxC ANL acZ, a new vector, as an i example below. this

Ax C ANL ac Z is expressed by a foreign gene (L ac Z) —CAG used for universal expression in transgenic mice to identify galactosidase (β-ga 1) activity Includes nuclear-targeted LacZ gene under the control of the promoter. The preparation of this vector can be performed as follows. For example,

Expression of foreign gene without producing E1A protein required for adenovirus replication by Kanegae et al.'S method (Kanegae, Y. et al., Ucleic Acids Research, 1995, vol. 23, 3816-3821) It can be prepared by inserting a LacZ gene derived from Escherichia coli under the control of the CAG promoter so that expression in cells and tissues can be confirmed.

The foreign gene used in the present invention is not particularly limited. From the viewpoint of usefulness, nucleotides having a base sequence encoding a bioactive protein, an antibody, a chromogenic protein as a marker gene, a drug-resistant protein as a marker gene and the like can be mentioned. Examples of bioactive proteins include cytokins (eg, interleukin 1-1-15, interferon-1, or a, tumor necrosis factor or granulocyte colony stimulating factor, erythropoietin, growth hormone, insulin, Insulin-like growth hormone, etc.), neurotrophic factors, α-1 antitribcine, blood coagulation factor 8, blood coagulation factor 9 and α-galactosidase. Examples of the chromogenic protein as a marker gene include β-galactosidase, GFP (Green fluorescent protein) and the like. Examples of drug-resistant proteins as marker genes include aminoglycoside phosphotransferase and the like.

The method for producing the transgenic animal of the present invention can be performed as follows. In the present invention, a transgenic animal refers to a non-human mammal containing a foreign gene introduced into the reproductive system. As such animal mouse, rat, Usagi, guinea pig, catcher formic, Hijji, pigs, mice below _c where © sheet etc. As an example, illustrates an exemplary method of creating Bok Ransuji Nick animals I do.

 First, superovulation is induced by intraperitoneally injecting PMSG (human chorionic gonadotropin) and hCG (human chorionic gonadotropin) into female mice. Use male mice of the same strain as the males for sperm donation. All cultures and reactions with the virus are performed at 37 ° C, 5% CO,, 95% air.

 } Sixteen hours after the administration of 1:10, unfertilized eggs wrapped in cumulus cells were treated with TYH (mouse in vitro fertilization medium (Toyoda et al., Jpn. J. Anim. Reprod. 16, 147-151). (1971))), spermatozoa pre-cultured for 2 hours in TYH are added so that the final concentration is 150 sperm / L, and in vitro fertilization is performed. Three to six hours after fertilization, the fertilized eggs were transferred to WM-EDTA (Medium for fertilized egg development (Hoshi et al., Jpn. J. Zootech. Sci. 56, 931-937 (1985))). Remove cumulus cells and egg membrane with Zelo and acidic Tyrode's solution (pH 2.5).

Next, non-proliferating adenoviruses, such as AxCANL ac Z, into which foreign genes have been introduced. The virus is infected at a dose of 10 to 10 ^a pfu Zm 1, preferably 5 × 10 ⁷ to 10 ″ pfu / m 1, and the reaction time with the virus is 2 to 8 hours. The infected embryo is transferred to the uterus of the parent mouse.

 Confirm whether the non-replicating adenovirus with the foreign gene introduced into the genome of the obtained offspring has been transmitted.

 Next, in the progeny F1 obtained by crossing the transgenic mouse obtained as described above with a wild-type mouse, the transgene is stably transmitted, and the recombinant non-proliferating adenovirus Confirm that the derived foreign gene is expressed. The following properties are observed in the transgenic mouse obtained as described above.

 As shown in the Examples below, one copy of integration, ie, complete adenovirus DNA, is observed in all three mice (FIG. 1). In contrast, when microinjecting DNA into the pronucleus of an egg, integration into transgenic animals occurs as a cluster of multiple transgenes arranged in a tandem head-to-tail fashion. These phenomena may indicate that the adenovirus terminal protein, unlike the naked DNA molecule, blocks the viral genome from cascading in mouse ova.

 Stable germline inheritance according to Mendel's law is observed, indicating that integration of the adenovirus genome occurred before or during DNA synthesis at the 1-cell stage. It has been reported that in the case of DNA microdigestion into the pronucleus, gene integration into fertilized eggs at the 1-cell stage occurs at a frequency of about 0% (Bi shop et al. Mol. Biol. Med., 6, 283-298 (1989)). In other cases of retroviral vectors, when infecting dividing embryos (preferably at the 4- or 8-cell stage), (Rubenstein et al., Proc. Natl. Acad. Sci.

U. S. A. 83, 366-368 (1986)), proviral integration can occur independently in one or more cells. Therefore, germline heritability is generally low due to mosaicism (Jaenisch et al., Cell 24, 519 529 (1981)).

In the case of inheritance of the adenovirus transgene in F1 progeny, no translocation is shown (Figure 3). On the contrary, it induces virus replication and cytotoxicity to host cells. Integration events in the case of wild-type adenoviruses containing the E1A gene that governs conduction appear to result in many translocations in cultured cell lines (Dorsch, MK et al., J. Virol. 34 , 305-314 (1980), Visser, L. et al., J. Virol. 39, 684-693 (1981)). One possibility is that the deletion of the cytotoxic E1A gene allows the integrated adenovirus DNA force to be maintained in the genome from one mouse cell to the germline in a stable state in the genome. It is.

 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

 Unless otherwise specified, the procedures for handling cosmids, DNA, various enzymes, Escherichia coli, and cultured cells and Southern analysis in the Examples are described in “Molecular Cloning A Laboratory Manual. T. Maniatis et al., 2nd edition. (1989), Cold Spring Harbor Laboratory.

Example 1

 Confirmation of transcription and translation in the oocyte pronucleus of the recombinant adenovirus genome

 First, we tested the ability of Ax CANLac Z to infect pronuclear single-cell ova in the presence or absence of the egg membrane.

One-cell eggs of mice were infected with 1 × 10 ⁸ pfu / ml of Ax CANL ac Z (an adenovirus having a nuclear translocation signal) with or without the egg membrane. The infected eggs were cultured and stained at the 2-cell stage 24 hours after infection. —Galactosidase activity was detected by histochemical (X—Ga1) staining (Beddington et al., Development 106, 37-46 (1989)).

 As a result, it was found that β-ga1 activity was clearly localized in the nuclei of 2-cell embryos (24 hours after virus infection) developed from infected eggs. The results indicated that the recombinant adenovirus genome translocated into the pronucleus of the ovum and was transcribed Z translated within 24 hours after infection. On the other hand, no / 3-gal activity was detected in the presence of the egg membrane. Therefore, removal of the egg membrane is a prerequisite for infection.

Example 2 Determining Whether the Adenovirus Genome Was Incorporated into the Mouse Egg Genome To do so, infected embryos in b stocyst (4 days after culture) were implanted into the uterus of the borrowing parent. Genomic DNA isolated from tail vein blood of 27 born mice was digested with restriction enzymes Eco RV and Hind III. Southern blot analysis revealed that three of the animals contained adenovirus DNA, as shown in Figure 1. Each of lanes 2 to 4 was identified as an internal fragment of the virus. Then, asterisks are put on putative fragments of the virus-one-cell DNA generated by integration of the adenovirus genome into the host genome.

 All three mice (ACL9.!, 9.3 and 9.23) carried one adenovirus genome, and the above analysis indicated that the adenovirus genome was randomly integrated into the mouse genome.

 experimental method

 Ec mouse genomic DNA (10 g). Shaku or! After digestion with indill, electrophoresis was performed on a 1.0% agarose gel, blotted on a nylon membrane Hybond-1N (manufactured by Amersham), and hybridized with a randomly digoxigenin-labeled probe, followed by Southern analysis.

Example 3

 Foreign genes in transgenic animals; expression of LacZ

 In addition, we tested the expression of the foreign gene LacZ in these three transgenic animals. Tissue pieces collected by punching the ears were stained by the X-ga1 staining method (Beddington et al., Development 106, 37-46 (1989)) in a conventional manner. Indeed — g a 1 activity was detected in two of the three transgenic animals (ACLs 9.3 and 9.23). However, it was detected in the other one (ACL9.1)

Example 4

 Transmission of recombinant adenovirus to progeny F1

To determine whether the adenovirus DNA had transmitted to progeny F1, three transgenic mice were naturally mated with ICR mice. Genomic DNA isolated from one F1 placenta on day 13.5 of gestation from one of the transgenic mice (FIG. 1, ACL9.23) was digested with HindIII. For Southern analysis Thus, it was found that the adenovirus transgene (ansgene) transmitted to 6 out of 10 F1 offspring (FIG. 3). These fragment patterns (FIG. 3, lanes 1, 3, 4, 7, 8, and 10) were consistent with the fragment patterns of the parental transgenic mice (FIG. 3, lanes ACL 9.23). The viral transgenes of the other two transgenic mice (ACL9.1 and AC L9.3) also transmitted to progeny F1 (11 out of 2 and 7 out of 13, respectively) ). Therefore, it can be said that the adenovirus transgene was confirmed to be stably inherited in F1 progeny. The transfer of the adenovirus transgene to the germ line was according to Mendel's law, and no translocation was observed (Figure 3).

Example 5

 Confirmation of transfection expression

 To further examine the expression of the adenovirus transgene in F1 progeny, 10 F1 fetuses from the same sample (FIG. 3) were stained for S—ga1 activity. Complete and intense dark blue staining was observed in 6 of the 10 F1 fetuses. This indicates that the inherited adenovirus transgene did indeed express the LacZ gene. In contrast, the other four fetuses that did not receive the adenovirus transgene did not stain at all. With respect to other transgenic mouse lines, 7 out of 13 F1 fetuses derived from ACL9.3 showed / 3-g a1 activity. Taken together, these data demonstrate that LacZ expression in F1 progeny resulted from stable transmission of the adenovirus transgene.

 experimental method

Whole fetuses were stained with X-ga1 to examine / 3-ga1 activity by a modification of the method of Allen et al. (Allen, ND et al., Nature 333, 852-855 (1988)). Briefly, fetuses were placed in phosphate buffer (pH 7.7) containing 0.5% formaldehyde, 2.5% glutaraldehyde, and 0.05% NP40 for 30 minutes at 4 ° C. Dipped and fixed. A second fixation was performed for another 15 minutes. The fixed fetuses were washed three times with a phosphate buffer, and stained with a staining solution (5 mM potassium fluoride, 5 mM potassium fluoride, 0.1% NP40 and 0.5 mgZ). Example 6: Incubation at 37 ° C for 4 hours in lϋmI phosphate buffer containing X-ga1 _c

 Confirmation of one copy of integrated adenovirus transgene In all three mice, one copy of integration, ie, complete adenovirus DNA, was observed (FIG. 1). In contrast, when DNA is injected by mouth into the pronucleus of the ovum, integration into the transgenic animal will result in multiple transgenes arranged in a tandem head-to-tail fashion. It occurs as a cluster. These phenomena may indicate that the terminal protein of the adenovirus, unlike the naked DNA molecule, prevents tandem formation of the viral genome in mouse ova.

Example 7

 Effect of virus administration on incorporation efficiency

 After removing the cumulus cells and egg membrane of the fertilized egg, use the dosages listed in Table 1.

Ax CANL ac Z was infected, cultured to b1 astocyst in vitro, and then returned to the borrower's uterus. Indeed, transplanted infected embryos ^{1 X 1 0 7, 5 X} 1 0 7 and ^{1 X 1 0 8 (pfu /} m 1), 5 5, 3 2, and 3 6 animals individuals respectively obtained Was. Table 1 shows the results of Southern blot analysis of these individuals using the LacZ gene as a probe. As apparent from Table 1, the dose of Wirusu as compared with the case of the ^{1 X 1 0 7 pfu / m} 1 (2 Z 5 5), when the ^{5 x 1 0 7 pfu / m} 1 (4Z3 2) and for ^{ix 1 0 8 pfu / m 1} (4/3 6) it can be seen that incorporation efficiency is remarkably improved. This is because, unlike normal virus infection, it is not necessary to consider the killing of animal cells due to an increase in virus dose, so that a higher dose of virus improves efficiency. It is. Table 1: Effect of virus dose on integration efficiency

 N D: Not measured

Industrial applicability

 According to the method of the present invention, transgenic animals can be easily and efficiently produced.

Claims

The scope of the claims 1. A transgenic animal characterized by transfecting a non-propagating recombinant adenovirus into which a foreign gene has been introduced into a fertilized animal egg from which the egg membrane has been removed, and then transplanting the embryo into the uterus of a parent animal. How to create  2. The transgene according to claim 1, wherein the foreign gene is a nucleotide having a nucleotide sequence encoding a bioactive protein, an antibody, a chromogenic protein as a marker gene, or a drug-resistant protein as a marker gene. Animal breeding method. 3. Non Dosage for growth recombinant adenovirus to infect animals fertilized removing the egg membrane is ^{5 xi 0 7 ~1 0 B pfu} Zm claims, characterized in that a l 1 or 2 The method for producing the transgenic animal described above.  4. The method for producing a transgenic animal according to any one of claims 1 to 3, wherein the non-propagating recombinant adenovirus is expressed under the control of the CAG promoter.  5. The method for producing a transgenic animal according to any one of claims 1 to 4, wherein the non-propagating recombinant adenovirus contains a nuclear transport signal.  6. Claims characterized in that only copies of the non-replicating recombinant adenovirus into which the foreign gene has been introduced are incorporated into the genome of the animal fertilized egg! 6. The method for producing a transgenic animal according to any one of items 5 to 5.  7. Transgenic animals created by infecting a fertilized animal egg from which the egg membrane has been removed with a non-replicating recombinant adenovirus into which a foreign gene has been introduced, and then transplanting the embryo into the uterus of a borrowing animal .