WO1997017843A1

WO1997017843A1 - Process for constructing transgenic animals

Info

Publication number: WO1997017843A1
Application number: PCT/JP1996/003359
Authority: WO
Inventors: Ryuichi Morishita; Yasushi Kaneda; Toshio Ogiwara; Tsuneaki Sakata; Mamoru Hasegawa
Original assignee: Dnavec Research Inc.
Priority date: 1995-11-16
Filing date: 1996-11-15
Publication date: 1997-05-22
Also published as: AU7587996A

Abstract

A process for easily constructing transgenic mammals comprising injecting into the amnion fluid of a mammal a composition for gene introduction containing a compound which has affinities for both DNA and the embryo of the mammal or the fetal body surface thereof and a vector for gene introduction. This process enables stage-specific gene introduction at various stages of the development or site-specific gene introduction which cannot be achieved by the conventional techniques.

Description

Description Method for producing transgenic animals

Technical field

The present invention relates to a technique for producing transgenic animals. Technology background

Regarding the method of creating a transgenic animal in which a foreign gene is introduced and stably expressed in vivo, a method of introducing a foreign gene into a fertilized egg of a mouse using a micromanipulator in 1980 was described by Gordon in the United States. First developed. In developmental biology and molecular biology, techniques for creating transgenic animals include: (1) analysis of the expression of genetic information that accompanies temporal changes such as gene development, differentiation, growth, and aging in vivo; Analysis of the gene structure involved in the expression of tissue-specific genetic information, ③ Analysis of the physiological and pathological roles of gene products at the individual level, 作成 Creation of disease model animals, ⑤ Occurred by the introduction of foreign genes It has been applied to the analysis of unknown genes using insertion mutations, etc., and 評価 the direct function evaluation of unknown genes. In addition, by creating a knockout animal in which a specific gene is inactivated by using a technology obtained by applying and developing this technology, it is also possible to identify the role of the gene at the developmental stage or the function in vivo. it can. Thus, transgenic technology has greatly contributed to the development of biotechnology. In addition, by applying this technology to livestock, it can be used to produce useful bioactive substances and improve varieties.

By the way, the most commonly used method for producing transgenic animals by a conventional method is to inject the isolated gene into the pronucleus of a fertilized egg through a glass capillary attached to the tip of a micromanipulator. I have. After injection, survived recipients The sperm is transplanted into the oviduct of the foster mother mouse. In other words, basically, three operations are performed: collection of a fertilized egg, injection of a gene, and transplantation of a fertilized egg into a fallopian tube. These operations require a high level of skill. Further, the number of transgenic mice finally obtained by the conventional method is about 1 to 3 on average when starting from 100 fertilized eggs, and the efficiency is very low. Furthermore, since the conventional method uses a fertilized egg, there is a disadvantage that it is impossible to knock out essential genes involved in the early stage of development.

Recently, the envelope protein of the Moroni mouse retrovirus vector and erythropoietin, which is one of the hematopoietic factors, are made into a fusion protein to appear on the virus surface, and the gene should be introduced specifically to erythroblasts. Kuta is also being developed (N. Kashihara et al., Science 266, 1373 1376 (1994)), but it has few successes and must be said to be technically immature. In particular, tissue-specific gene transfer is impossible with conventional transgenic technology, and there is no conceptually described reference to gene transfer specific to “whole body surface”. Disclosure of the invention

An object of the present invention is to overcome the technical difficulties of the conventional technology and to easily produce a transgenic mammal. It is another object of the present invention to introduce a gene in a stage-specific and site-specific manner at various stages of development, which was impossible with the prior art.

The present inventors have found that a composition containing a compound having an affinity for both DNA and cells and a vector for gene transfer can be efficiently contacted with the epidermis of a mammal including living epidermis cells directly. The present inventors have found that gene transfer can be performed and completed the present invention. More specifically, the present inventors have found that, when a gene transfer vector is introduced into fetal amniotic fluid, the gene is transferred in a body surface-specific manner over the entire body surface, thus completing the present invention. did.

That is, the present invention provides a method for producing a transgenic mammal other than a human, Regarding the composition for gene transfer used in the method, specifically,

(1) In the mammalian amniotic fluid, in order to specifically transfer a foreign gene across the entire body surface of a mammalian embryo or fetus, DNA and both the DNA and the embryo or fetus body surface of the mammal A method for producing a transgenic mammal other than a human, which comprises injecting a gene transfer composition comprising a compound having affinity and a gene transfer vector.

(2) The method according to (1), wherein the compound having an affinity for both the DNA and the mammalian embryo or fetal body forms ribosomes.

(3) the method according to (1) or (2), wherein the composition for gene transfer contains an inactivated Sendai virus (HV J);

(4) A composition for gene transfer, which is used in the method according to any one of (1) to (3),

About.

Examples of the “compound having an affinity for both DNA and the body surface of a mammalian embryo or fetus” in the “gene transfer composition” of the present invention include cationic ribosomes (PLFelgner et al., Pro Natl. Acad. Sci. USA, 84, 7413 (1987), CM Corma et al., Ol. Cell. Biol., 2, 1044 (1982), N. Haga, K. Yagi, J. Clin. Biochem. Nutr. , 7, 175 (1989), JR Neuian et al., Biotechniques, 12, 643 (1987)), liposomes containing HVJ-ribosomes (“Ribosomes in Life Sciences / Experimental Manual”, Springer Phuarark Tokyo (1992) pp. 282 to 287). As the “gene transfer vector” in the “gene transfer composition” of the present invention, basically all gene transfer vectors are used. For example, retrovirus vectors, adenovirus vectors Virus vectors for gene transfer such as adeno-associated virus vectors.

As a method for injecting the “gene introduction composition” of the present invention into amniotic fluid, there is a method of injecting directly into amniotic fluid in the uterus using an injector or the like. Further, as the genes to be guided by the “gene transfer vector” of the present invention, all types of genes such as development stage-specific genes, organ-specific, particularly skin-specific shoalers, and other genes for treating various diseases are included. No.

Furthermore, the range of animals to which the method of the present invention can be applied or the range of animals to which the composition of the present invention can be administered includes all mammals such as mice, puppies, puppies, and monkeys except humans. It is possible.

According to the present invention, compared to the prior art of introducing a gene into a fertilized egg, the fertilized egg which is a pre-implantation embryo for embryo development, as well as the 2-cell stage embryo, the 4-cell stage embryo, and the 8-cell stage Genes can be introduced into embryos at any stage, including embryos, morulae, blastocysts, late blastocysts, early ovarian embryos, ovarian embryos, and late ovarian embryos. The gene can also be introduced into the embryo after implantation. Such "introduction of a stage-specific gene" according to the present invention is considered to give very important findings to embryology research. In particular, the role of the gene at each stage of development can be inferred by suppressing genes that act stage-specifically, such as by introducing antisense. In addition, by introducing the gene into each site at each developmental time, the future fate of the development at that site and the fate of the cell can be observed. Furthermore, even if the gene is not convenient to act on all tissue organs, introduction of the gene after a certain stage of development can avoid toxicity due to overexpression of the gene. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 (A)-Fig. 1 (B) are photomicrographs showing the distribution of FITC-labeled 0DN introduced into fetal rat amniotic fluid by IS-injector after 2 hours. (A) is a micrograph observed at 40 × and (B) is a micrograph observed at 100 ×.

Figure 2 is a photomicrograph showing the distribution of FITC-labeled 0DN 5 days after introduction into fetal rat amniotic fluid by direct injection.

Figure 3 shows the structure of plasmid pSV40Gal expressing ^ -galactosidase gene. FIG.

FIG. 4 (A) -FIG. 4 (C) are photographs of rat fetal skin on day 5 after transfusion. (A) and (B) relate to rats transfected with the S-galactosidase vector and control vector, respectively, and (C) relates to untreated rats.

FIG. 5 (A) -FIG. 5 (C) are photographs showing histological analysis of rats on day 5 after transfusion. (A) and (B) relate to a rat transfected with one galactosidase vector and a control vector, respectively, and (C) relates to an untreated rat.

FIG. 6 (A) -FIG. 6 (C) are photographs showing histological analysis of rats 10 days after transfection. (A) and (B) relate to the rat transfected with the -galactosidase vector and the control vector, respectively, and (C) relates to the untreated rat. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Preparation of HVJ-liposome

Phosphatidylserine, phosphatidylcholine, and cholesterol were mixed at a weight ratio of 1: 4.8: 2. The dried lipid was dissolved in 200 1 BSS solution (137 mM NaCl, 5.4 mM C1, 10 mM Tris-HCl, pH 7.6) containing plasmid DNA or FITC-labeled oligonucleotide (0DN). The ribosome was prepared by shaking and ultrasound (see “Liposome Experiments in Life Sciences”, Springer 1. Pharaak Tokyo (1992), p. 282-287).

On the other hand, HVJ (strain Z) was purified and inactivated by UV irradiation (110 erg / nun ² / sec) for 3 minutes immediately before use. The ribosome solution described above (10 mg liposome / 0.5 ml solution) was mixed with 10,000 hemagglutination units of HVJ and diluted with BSS solution to a final volume of 4 ml. The mixed solution was kept at 4 for 5 minutes, and then gently shaken at 37 for 30 minutes. Unreacted HVJ and liposomes were removed from the HVJ-ribosome by a sucrose density gradient. [Example 2] In vivo introduction of FITC-labeled oligonucleotide (0DN)

The oligonucleotide (0DN) was labeled with FITC at the 5 'end and 3' end (Antisense Res. Develop. Vol. 2, 27-39 (1992)). The HVJ-ribosome containing the labeled ODN was prepared by the method of Example 1. The final concentration of the FITC-labeled 0DN injected into the amniotic fluid was 3 M (concentration of 0DN relative to the total ribosome solution). To the amniotic fluid of the Sprague-Dawley rat on the 15th day of pregnancy, 101 μl of HVJ-ribosome containing labeled 0DN prepared by the method of Example 1 was injected through a 30-gauge injection needle. . At this time, the injector was shot directly into the amniotic fluid from the uterine wall. As a control, HVJ ribosomes containing no FITC-labeled 0DN prepared by the method of Example 1 were injected. All rats were sacrificed 2 hours or 5 days later, and the extracted tissues were fixed in a 4% paraformaldehyde solution according to a conventional method, and the sections were stained with erythrochrome black and observed with a fluorescence microscope.

As a result, fluorescence was observed in several layers of epidermis and epithelium throughout the whole body 2 hours after the introduction (FIGS. 1 (A) and 1 (B)). No fluorescence was observed in rats injected with HVJ ribosomes without FITC-labeled 0DN or uninjected (data not shown). Epidermal cell fluorescence was mainly observed in the nucleus and was observed for at least 5 days after transfection (Fig. 2). Although the fluorescence was low, it was observed in the liver, but not in the kidney, heart, lung, and brain.

[Example 3] Introduction of; 3-galactosidase into neonatal rat

pSV40Gal, a plasmid that expresses the β-peroxidase gene, was constructed as follows. Plasmid DNA pAct-c-myb containing the 370 base pair 5 'promoter overnight region and the 900 base pair primary 1-xon region of the nitrile / S-actin promoter to remove the cmb region Cleavage with restriction enzymes Ncol and Xbal was followed by ligation with Sail linker. 3. One kilobase pair of E. coli; the 3-galactosidase gene was excised from the plasmid DN ApMC1871 using the restriction enzyme Sail and bound to the Sail site on the plasmid thigh (Fig. 3). As a control, a plasmid DNA pAct-CV containing no E. coli yS-galactosidase gene was simultaneously prepared. HVJ-ribosomes containing the 9-galactosidase gene plasmid pSV40Gal or control plasmid DNA pAct-CV were prepared. To increase transfection efficiency, HMG-1 protein was simultaneously encapsulated in HVJ-ribosomes. A syringe was injected directly into the amniotic fluid from the uterine wall, and 10〃1 of HVJ-ribosome prepared by the method of Example 2 was injected. Rats were sacrificed 5 days and 10 days after injection, and the extracted tissue was placed in a 1% paraformaldehyde solution and fixed according to a conventional method, and the tissue was removed. Staining solution. Then, a solution of X gal chromadin buffer (49 mg / ml 5 bromo-4-chloro-3-indolyl D-galactoside: 1 mmol MgCl ₂ and 3 ol K ₃ Fe (CN) ₆ dissolved in PBS = l: 99). After staining, the cells were observed with a microscope. As a control, the same operation was performed on rat hearts into which HVJ liposomes containing plasmid DM containing no Escherichia coli-galactosidase gene had been injected.

As a result, expression of yS-galactosidase was observed in fetal skin after 5 days by directly injecting HVJ-liposome containing the 3-galactosidase gene plasmid DNA vector into amniotic fluid (Fig. 4 (A)). On the other hand, no expression of -galactosidase was observed in HVJ-liposomes containing no> 9-galactosidase gene plasmid DNA vector or untreated fetal skin (Fig. 4 (B), Fig. 4 ( C)). Cells in which S-galactosidase expression was observed were present in the epidermis and several layers of epithelium (Fig. 5 (A)). These cells were mainly fibroblasts. The expression of S-galactosidase was observed over 10 days, although the expression level was weak (Fig. 6 (A)). On the other hand, no galactosidase expression was observed in the tissues of HVJ-ribosome-treated mice that did not contain the / S-galactosidase gene plasmid DNA vector and untreated mice (Fig. 5 (B) (Fig. 5 (C), Fig. 6 (B), Fig. 6 (C)) Note that there was no difference in the survival rate between the rat into which HVJ-ribosome was introduced and the rat into which HVJ-ribosome was not introduced. Industrial applicability

According to the present invention, it becomes possible to introduce a gene specifically to “the whole body surface”. However, transgenic animals can be created more easily than in the past. Transgenic animals in which genes have been specifically introduced into the entire body are particularly useful for studies of skin cells, including fibroblasts. Specifically, (1) the expression of genetic information associated with temporal changes such as the development, differentiation, growth, and aging of skin-specific genes; (2) the analysis of the gene structure involved in the expression of skin-specific genetic information; and (3) the skin. It can be applied to research such as creation of disease model animals. In addition, the present invention can be applied to the production of useful livestock with altered skin characteristics. In addition, using the method of the present invention, knockout of essential genes and introduction of lethal genes have become possible, and the range of transgenic animals that can be produced has been expanded. Furthermore, “stage-specific gene transfer” has become possible using the present invention.

Claims

The scope of the claims

1. In mammals' amniotic fluid, the affinity for both the DNA and the mammalian embryo or fetal body surface to transfer the foreign gene specifically throughout the mammalian embryo or fetal body surface. A method for producing a transgenic mammal other than a human, comprising a step of injecting a gene transfer composition containing a compound having the compound and a gene transfer vector.

2. The method according to claim 1, wherein the compound having an affinity for both the DNA and the mammalian embryo or fetal body forms ribosomes.

3. The method according to claim 1 or 2, wherein the composition for gene transfer comprises Sendai virus (HVJ) inactivated.

4. A composition for introducing a gene, which is used in the method according to any one of claims 1 to 3.