WO2023282552A1 - Production of cognitive impairment model dog expressing human mutant presenilin-1 - Google Patents

Abstract

The present invention relates to production of Alzheimer's disease (AD) model dog clone by using a somatic cell nuclear transfer technique. More specifically, the present invention relates to development of cells overexpressing a human PS1 mutant gene, which is one of the etiological genes of human AD, and production of a PS1-expressing transgenic dog by using same.

Description

Production of HUMAN MUTANT PRESENILIN-1 expression model for cognitive impairment

The present invention relates to the production of an Alzheimer's disease (AD) model cloned dog using a somatic cell nuclear transfer technique. More specifically, it relates to the development of cells in which the human PS1 mutant gene, one of the human AD pathogenesis genes, is overexpressed, and the production of transgenic dogs expressing PS1 using the same.

BACKGROUND OF THE INVENTION Modern society has entered a rapidly aging society due to the development of medicine, and as a result, the incidence of degenerative cranial nerve diseases resulting in cognitive impairment is increasing. Alzheimer's disease (AD), known as a representative degenerative neurological disease of cognitive impairment, begins with brain function deterioration as neurotoxic substances such as amyloid, an abnormally aggregated protein mass, accumulate in the temporal lobes of both sides, and gradually develop in the brain's cortex. The disease progresses as the accumulation of amyloid increases inside and outside the nerve cells of the part. The main symptoms begin with memory impairment, cause language disorders, etc., lose the ability to think or reason, and eventually lead to death. AD shows a tendency to increase in incidence with age, and occurs mainly in the population over 60 years of age, but recently, it is not uncommon to occur in young people in their 30s.

In order to solve this problem, various animal models have been made and efforts have been made to study the disease for a long time. Mice have been widely used as experimental animals based on their short breeding cycle, rapid generation change, and ease of handling, but due to differences in breeding and physiology with humans, it has been difficult to apply clinical results directly to humans. It is true. Therefore, efforts are currently underway to develop animal models with pathophysiology close to humans.

In addition, since most of the experimental animal models are aimed at modeling the symptoms and pathological causes of each disease, it is most important to make the pathophysiology identical to that of humans. Even in AD mouse models produced to date, most of them produce models in which only partial lesions appear. As for the production methods of these models, direct drug injection into the mouse brain and pronuclear microinjection of the lesion gene into the pronuclear microinjection are mainly used to produce disease model mice. However, although this method can produce disease model animals with the animals we want, it shows problems of low production efficiency (cattle 0.5%, pig 1.5%, sheep 2.5%) and mosaicism. Therefore, many researchers are currently attempting to produce transgenic experimental animals with the same genetic traits with higher efficiency by applying the somatic cell cloning technique and the transformation technique together.

On the other hand, dogs are in the spotlight as next-generation disease model animals because they are genetically close to humans and have many important genetic diseases similar to humans. According to Webb's OMIA, in the case of dogs, there are about 300 disease models that can be used for human diseases, showing many disease patterns similar to humans after rodents. And as a result of years of searching for animals that spontaneously cause diseases similar to those of AD, dogs have been reported as representative animals that naturally have plaques in the brain. Therefore, if the AD model cloned dog is produced using a dog as in this study rather than any other animal, it will provide great benefits in the diagnostic method and causal study of AD.

One object of the present invention is to provide a transgenic dog expressing human PSEN1 (presenilin-1).

Another object of the present invention is to provide a method for producing transgenic dogs expressing human PSEN1 (presenilin-1).

Another object of the present invention is to provide a neural-specific PSEN1 expression vector.

The present invention provides a transgenic dog expressing human PSEN1 (presenilin-1).

The transgenic dog may have a genome including the human PSEN1 gene.

The human PSEN1 gene may be a gene encoding a presenilin-1 mutant protein.

In this case, the presenilin-1 mutant protein may be a mutation related to Alzheimer's disease.

In this case, the presenilin-1 mutant protein may be I416T mutation, A79V mutation, H163R mutation, F388L mutation, I143T mutation, L381V mutation, C410Y modified mutation, L286V mutation, L435F mutation or M146V mutation. Preferably, the presenilin-1 mutant protein may be M146V mutant.

The human PSEN1 gene may be operably linked to a synapsin (SYN) promoter.

In this case, the human PSEN1 gene may be specifically expressed in nerve tissue.

The transgenic dog may be an animal model for cognitive impairment.

In this case, the cognitive impairment may be Alzheimer's disease (AD).

The present invention provides a method for producing a transgenic dog expressing human PSEN1 (presenilin-1) comprising:

(a) preparing nuclear donor cells comprising culturing somatic cells or stem cells isolated from dogs;

(b) introducing a recombinant vector containing a SYN promoter and a human PSEN1 gene into the nuclear donor cell;

(c) removing nuclei from dog eggs to produce enucleated eggs;

(d) microinjecting and fusing the nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); and

(f) implanting the activated eggs into the oviduct of the surrogate mother.

The human PSEN1 gene may be a gene encoding a presenilin-1 mutant protein.

In this case, the presenilin-1 mutant protein may be a mutation related to Alzheimer's disease.

In this case, the presenilin-1 mutant protein may be I416T mutation, A79V mutation, H163R mutation, F388L mutation, I143T mutation, L381V mutation, C410Y modified mutation, L286V mutation, L435F mutation or M146V mutation. Preferably, the presenilin-1 mutant protein may be M146V mutant.

The transgenic dog may be a cognitive disorder model animal.

In this case, the cognitive impairment may be Alzheimer's disease (AD).

The present invention provides a neural-specific PSEN1 expression vector.

The vector may include a human PSEN1 gene operably linked to a synapsin (SYN) promoter.

The human PSEN1 gene may be a gene encoding a presenilin-1 mutant protein.

In this case, the presenilin-1 mutant protein may be a mutation related to Alzheimer's disease.

In this case, the presenilin-1 mutant protein may be I416T mutation, A79V mutation, H163R mutation, F388L mutation, I143T mutation, L381V mutation, C410Y modified mutation, L286V mutation, L435F mutation or M146V mutation. Preferably, the presenilin-1 mutant protein may be M146V mutant.

The vector may be a plasmid, a transfer factor-containing vector, a viral vector, or other media known in the art. Preferably, it may be a transfer factor-containing vector or a viral vector.

In this case, the viral vector may be a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a vaccinia virus vector, a poxvirus vector, or a herpes simplex virus vector.

The production of the transgenic cloned dog (PS1 model dog) of the present invention establishes a foundation for the production of Alzheimer's disease model dog, and at the same time, the possibility of producing cloned dogs of other disease models can be verified. In addition, the production of PS1 model dogs can enable mass production of disease model animals due to the fecundity of dogs.

Figure 1 shows the structure of the neuron-specific mutant PS1 expression vector.

Figure 2 is a graph confirming the immunophenotype of adipose stem cells using a flow cytometer.

Figure 3 shows the human PS1M146V mutation nucleotide sequence.

4 is a genetic verification result of PS1 puppy expressing mutPS1MK16V- Green gene. At this time, A) is the PCR analysis result using the blood of PS1 puppy, and B) is the PCR analysis result using the blood of PS1-1 puppy. Table C) is a table summarizing the primers used in the analysis. (P: pPB-hScyn1p-mutPS1(M416V)-Green-puro vector; N: same-age common beagle; TG: transgenic clone dog PS1 or PS1-1)

Figure 5 is the result of Southern blot analysis confirming transgene integration of PS1 puppy. At this time, A) is the Southern blot analysis result of confirming transgene integration of PS1 puppy by 2 cuts using HindIII and SacI, and B) is the Southern blot analysis result of confirming transgene integration of PS1 puppy by 1 cut using HindⅢ. (P: pPB-hScyn1p-mutPS1(M416V)-Green-puro vector; N: Normal beagle of the same age; TG: transgenic clone dog PS1)

6 is a result of blood/serological health status examination of PSEN1-expressing beagle.

7 is a comparison analysis result of canine PSEN1 and human PSEN1 expression levels according to the experimental group through ELISA analysis. Significant differences between groups were marked as a, b, c (p < 0.05). K9_PSEN1: canine presenilin 1, human_PSEN1: human presenilin 1, Young: 3 beagles of the same age (mean ± SE), Old: 2 elderly dogs with cognitive impairment (mean ± SE), PSEN1: PSEN1 expression beagles (mean ± SE)

8 is a comparative analysis result of canine Tau expression concentration according to the experimental group through ELISA analysis. Significant differences between groups were marked as a and b (p < 0.05). K9_Tau: canine Tau, Young: 3 beagles of the same age (mean ± SE), Old: 2 elderly dogs with cognitive impairment (mean ± SE), PSEN1: PSEN1 expressing beagles (mean ± SE)

9 is a comparative analysis result of canine Tau expression level according to the experimental group through ELISA analysis. Significant differences between groups were marked with a, b, (p < 0.05). K9_APP: canine beta-amyloid precursor protein, Young: 3 beagles of the same age (mean ± SE), Old: 2 elderly dogs with cognitive impairment (mean ± SE), PSEN1: PSEN1 expressing beagles (mean ± SE)

10 shows measurement items of MRI images.

11 shows nine anatomical structures for DTI analysis. (Left), Corpus callosum genu region; (middle), Corpus callosum body region; (Right), Corpus callosum splenium region. 1. Internal capsule (anterior limb, right); 2. Internal capsule (anterior limb, left); 3.Corpus callosum (genu); 4. Internal capsule (genu, right); 5. Internal capsule (genu, left); 6. Corpus callosum (body); 7. Internal capsule (posterior limb, right); 8. Internal capsule (posterior limb, left); 9. Corpus callosum (splenium)

12 is a T1-weighted transverse image of the brain. No differences in cerebral sulci width (red arrows) were observed. Thicker epidural fat was observed in PSEN-1 dogs.

13 is a T1-weighted dorsal image, and no significant cortical atrophy was observed.

Figure 14 is a T2-weighted transverse image showing moderate ventricular hypertrophy in PSEN-1 dogs (brain height/lateral ventricular height = Rt. 0.20, Lt. 0.18). No signs of ventricular hypertrophy were observed in Ori-11 (brain height/lateral ventricular height). = Rt. 0.08, Lt. 0.11).

15 is a T2-weighted transverse image, showing no difference in thalamic adhesion length (Ori 11: 7.95 mm, PSEN-1: 7.63 mm).

16 is a T2-weighted transverse image, showing no difference in thalamic adhesion length (Ori 11: 7.95 mm, PSEN-1: 7.63 mm).

17 is a T2-weighted sagittal image plane, and no difference was observed in terms of adhesion morphology (arrow) between the thalamus.

18 is a FLAIR transverse image, showing no signs of high intensity around the ventricles of a PSEN-1 dog.

19 is an image showing DTI analysis of Corpus Callosum (body). Ori 11: MD (mean 0.000918), FA (mean 0.4935), PSEN-1: MD (mean 0.001505), FA (mean 0.2989)

20 is an image showing DTI analysis of the internal capsule.

21 is an image showing DTI analysis of the caudate nucleus.

22 is an image showing DTI analysis of Fornix.

23 is an image showing DTI analysis of Hippocampus. Ori 11: Rt. MD (mean 0.001045), Rt. FA (mean 0.5703), Lt. MD (average 0.001175), Lt. FA (average 0.2965), PSEN-1: Rt. MD (mean 0.002049), FA (mean 0.3370), Lt. MD (mean 0.002113), Lt. FA (average 0.3280)

The present invention relates to a transgenic dog expressing human PSEN1 (presenilin-1), wherein the transgenic dog has a genome including the human PSEN1 gene, wherein the human PSEN1 gene is a gene encoding a presenilin-1 mutant protein. It relates to transgenic dogs characterized by

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or identical to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definitions of representative terms used in the present invention are as follows.

A "vector" or "expression vector" is a plasmid known in the art, into which a nucleic acid encoding a gene (target gene) to be expressed can be inserted, and the nucleic acid can be expressed in a host cell. element) containing vector, viral vector or other carrier. Preferably, it may be a transfer factor-containing vector or a viral vector.

A "recombinant vector" is a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert.

"Expression control sequence" or "regulatory element" means a DNA sequence that controls the expression of an operably linked nucleic acid sequence in a particular host cell. Such regulatory sequences include any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating termination of transcription and translation. In the present invention, regulatory factors may be promoters, enhancers, and the like.

"Promoter" refers to a DNA sequence capable of controlling the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence. Usually, the promoter is present 5' (i.e., upstream) of the desired nucleotide sequence to be transcribed into mRNA, although not applied in all cases, and is a site where RNA polymerase and other transcription factors for initiation of transcription specifically bind. to provide. The promoters of the present invention are constitutive promoters. The term "constitutive," as used in reference to a promoter, means that the promoter is capable of directing the transcription of operably linked nucleic acid sequences without stimulation (eg, heat shock, chemicals, etc.). The promoter of the present invention may preferably be a neuron-specific promoter. The neuron-specific promoter can be regulated so that a specific sequence linked to the promoter is transcribed in a neuron-specific manner. The neuron-specific promoter may be a synapsin promoter.

"Operably linked" refers to functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest so as to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the encoding nucleic acid sequence. Operational linkages with recombinant vectors can be made using genetic recombination techniques well known in the art.

"Transformation" means to change the genetic properties of an organism by DNA given from the outside. Transformation methods include various methods known in the art, such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, and viral infection. Methods (viral infection), direct muscle injection (direct muscle injection), insulator (insulator), and techniques using transposon (transposon) can be appropriately selected and applied. Preferably, in the present invention, canine adipose stem cells or canine fetal cell lines can be transformed by introducing an expression vector containing the human PSEN1 (PS1) gene.

"Cell", "host cell", "modified host cell" and the like refer to a particular subject cell, as well as progeny or potential progeny of such a cell. Because certain modifications may occur in progeny by mutation or environmental influences, such progeny, in fact, will not be identical to the parent cell, but are still included within the term and scope as used herein.

The term "model animal for disease research" refers to animals having diseases very similar to human diseases or having disease resistance. In the study of human disease, disease model animals have significance due to physiological or genetic similarities between humans and animals. In disease research, biomedical disease research model animals provide materials for research on various causes, onset processes, and diagnosis of diseases, and through disease research model animal studies, disease-related genes are identified and interactions between genes are studied. In addition, through the actual efficacy and toxicity test of the developed new drug candidate, basic data can be obtained to determine the feasibility of commercialization.

"Animal" or "laboratory animal" means any mammalian animal other than human. The animals include animals of all ages including embryos, fetuses, neonates and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals include laboratory animals or other animals, rabbits, rodents (eg mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (eg chickens, turkeys, ducks, geese), primates (eg chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a dog.

"Nuclear transfer" refers to a genetic manipulation technology that artificially binds another cell or nucleus to a denuclearized egg to have the same trait. "Nuclear transfer embryo" refers to an egg into which a nuclear donor cell has been introduced or fused.

"Cloning" is a genetic manipulation technology that creates a new individual having the same set of genes as one individual, and in particular, in the present invention, somatic cells, embryonic cells, fetal-derived cells, adipose stem cells, and/or adult-derived cells of a dog are used to sequence the nuclear DNA sequences of other cells. It refers to having a nuclear DNA sequence substantially identical to that of The present invention uses a technique for cloning dogs using nuclear transfer technology. In particular, somatic cell nuclear transfer technology is a technology that allows the birth of offspring without going through meiosis and reproductive cells containing hemichromosomes, which are common in the reproductive process. It is a method of generating a new organism by producing and transplanting the fertilized egg into a living body.

"Nuclear donor cell" refers to a cell or nucleus of a cell that transfers a nucleus to a nuclear receptor, a recipient oocyte. "Oocyte" preferably refers to a mature egg that has reached the second meiosis metaphase. In the present invention, somatic cells or stem cells of dogs may be used as the nuclear donor cells.

A "somatic cell" is a cell other than a germ cell among cells constituting a multicellular organism, a differentiated cell that is specialized for a certain purpose and does not become a cell other than that, and the ability to differentiate into a cell having various other functions. contains cells with

"Stem cell" means a cell capable of developing into any tissue. There are two basic characteristics, first of all, self-renewal (self-renewal), which creates itself by repeated division, and has the ability to differentiate into cells with specific functions depending on the environment.

"Cultivation" refers to growing living organisms or parts of living organisms (organs, tissues, cells, etc.) under appropriately artificially controlled environmental conditions. Partial pressure) is important, and the medium (incubator) that has the most important direct effect on the organism being cultured is a direct environment for the organism and a supply of various nutrients necessary for survival or growth.

"In vitro culture" refers to a series of laboratory processes in which cells are cultured in an incubator in a laboratory under conditions similar to the environment in the body in a way that is distinct from the state in which cells are grown in the body.

"Medium" or "media composition" refers to a mixture for the growth and proliferation of cells in vitro, including essential elements such as sugars, amino acids, various nutrients, serum, growth factors, and minerals.

"Living offspring" refers to an animal capable of surviving outside the womb. Preferably, it refers to an animal that can survive for 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months or 1 year or more. The animal does not require an intrauterine environment for survival.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, disease state improvement or palliation and relief (partial or total), detectable or undetectable. “Treatment” refers to both therapeutic treatment and prophylactic or prophylactic methods. The treatments include treatment required for disorders that have already occurred as well as disorders that are prevented. "Palliating" a disease means reducing the extent and/or undesirable clinical signs of the disease state and/or slowing or prolonging the time course of the disease compared to no treatment. means to lose

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for a reference amount, level, value, number, frequency, percentage, measure, size, amount, weight or length means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by 3, 2 or 1%.

Throughout this specification, unless the context requires otherwise, the terms "comprises" and "comprises" include a given step or element, or group of steps or elements, but any other step or element, or step or element. It should be understood to imply that groups of are not excluded.

Hereinafter, the present invention will be described in detail.

One aspect of the content disclosed by this specification relates to transgenic animals expressing PSEN1 (PS1 or presenilin-1).

More specifically, it relates to a transgenic dog expressing nerve-specific PSEN1 (PS1), a method for producing the same, and a use thereof.

The nerve-specific PSEN1 (PS1) expression transgenic dog is characterized in that it is used after being transformed to specifically express the PSEN1 (PS1) protein in nerve tissue or cells constituting the nerve tissue.

At this time, PSEN1 (PS1) gene can be transformed using any known method, but preferably a viral vector is used. That is, a viral vector containing a synapsin (SYN) promoter and a specific gene PSEN1 (PS1) is introduced into somatic cells or stem cells, and the SYN promoter and PS1 gene are inserted into the genome to produce transformed cells or cell lines.

A neuron-specific PSEN1 (PS1)-expressing transgenic dog can be produced using the produced transformed cell or cell line, and at this time, a neuron-specific PSEN1 (PS1)-expressing transgenic dog can be produced using any known method. However, preferably, somatic cell nuclear transfer (SCNT) is used. Neuron-specific PSEN1 (PS1) expressing transgenic animals of the present invention were prepared using a somatic cell nuclear transfer (SCNT) method using a transgenic cell line into which the SYN promoter and PS1 gene were inserted in the genome, that is, a transgenic cell line in which PS1 was expressed. produce

Therefore, for example, the method for producing a neuron-specific PSEN1 (PS1) expressing transgenic dog of the present invention through a somatic cell nuclear transfer method,

(a) preparing nuclear donor cells comprising culturing somatic cells or stem cells isolated from dogs;

(b) introducing a recombinant vector containing a SYN promoter and a PSEN1 (PS1) gene into the nuclear donor cell;

(c) removing nuclei from dog eggs to produce enucleated eggs;

(d) microinjecting and fusing the nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); and

(f) implanting the activated eggs into the oviduct of the surrogate mother.

Conventional descriptions of each step will be understood by referring to methods for producing cloned animals using conventional somatic cell nuclear transfer technology known in the art.

[Nerve specific PSEN1 (PS1) expression system]

Neuron-specific PSEN1 (PS1) expression recombinant vector

The present disclosure provides a neural-specific PSEN1 (PS1) expression vector that specifically expresses PSEN1 (PS1) in neural tissue or cells constituting the neural tissue.

In particular, the vector is characterized by including a synapsin (SYN) promoter and a PSEN1 (PS1) gene. The synapsin (SYN) promoter and the PSEN1 (PS1) gene can be used in an operably linked form.

In this case, the PSEN1 (PS1) gene may be a wild type PSEN1 (PS1) gene.

At this time, the PSEN1 (PS1) gene may be a mutant PSEN1 (PS1) gene. The mutant PSEN1 (PS1) gene is a gene encoding a PSEN1 (PS1) mutant protein, and the PSEN1 (PS1) mutant protein is an I416T mutation (a mutant in which the 416th isoleucine is transformed into threonine), A79V (a 79th alanine is a valine mutation), H163R (mutation in which histidine at position 163 is transformed into arginine), F388L (mutation in which phenylalanine at position 388 is transformed into leucine), I143T (mutation in which isoleucine at position 143 is transformed into threonine), L381V (mutation in which position 381 is transformed into threonine) Leucine to valine mutation), C410Y (the 410th cysteine to tyrosine mutation), L286V (the 286th leucine to valine mutation), L435F (the 435th leucine to phenylalanine mutation) or M146V ( 146th methionine is modified to valine), etc., but is not limited thereto. The PSEN1 (PS1) mutant protein may be a mutation related to Alzheimer's disease.

The nerve-specific PSEN1 (PS1) expression vector is a plasmid known in the art, a vector containing a transfer factor, a viral vector, or a vector known in the art capable of specifically expressing a nucleic acid encoding the PS1 gene in nerve tissue or cells constituting the nerve tissue. As other mediators, it may preferably be a vector containing a transfer factor or a viral vector.

In one embodiment,

The vector may be a viral vector or a recombinant viral vector.

In this case, the virus may be a DNA virus or an RNA virus.

In this case, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

At this time, the RNA virus may be a single-stranded RNA (ssRNA) virus.

In this case, the virus may be retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus, but is not limited thereto.

In general, a virus may infect a host (eg, a cell) to introduce a nucleic acid encoding viral genetic information into the host or insert a nucleic acid encoding genetic information into the genome of the host. The PSEN1 (PS1) gene can be introduced into a target (eg, cell) using a virus having these characteristics. The PSEN1 (PS1) gene introduced using a virus can be transiently expressed in a subject (eg, a cell). Alternatively, the PSEN1 (PS1) gene introduced using a virus can be used in a subject (eg, cell) for a long period of time (eg, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months). months, 1 year, 2 years or permanently).

The packaging capacity of the virus may vary depending on the type of virus, ranging from at least 2 kb to 50 kb. Viral vectors containing the PSEN1 (PS1) gene and/or additional components can be designed according to this packaging capability.

For example, the nerve-specific PSEN1 (PS1) expression vector may be a lentiviral vector or a recombinant lentiviral vector.

As another example, the neural-specific PSEN1 (PS1) expression vector may be an adenoviral vector or a recombinant adenoviral vector.

In another embodiment,

The vector may be a vector containing one or more transfer factors.

The transfer factor may be a piggyBac transfer factor or a Sleeping Beauty transfer factor.

The transfer factor may include an inverted terminal repeat sequence (ITR) and/or a direct repeat (DR) at both ends.

Enzymes that mediate transposition of the transfer factor may include piggyBac transposase or Sleeping Beauty transposase.

In this case, the vector may be a piggyBac vector or a Sleeping Beauty vector.

The nerve-specific PSEN1 (PS1) expression vector may further include a selection marker, wherein the selection marker includes antibiotic resistance genes such as kanamycin resistance gene and neomycin resistance gene, and fluorescence such as green fluorescent protein and red fluorescent protein. proteins and the like, but are not limited thereto.

In addition, in order to confirm whether the PS1 protein is expressed, a tag sequence for protein separation or purification may be further included in the vector of the present invention. Examples of the tag sequence include GFP, GST (Glutathione S-transferase)-tag, HA, His-tag, Myc-tag, and T7-tag, but the tag sequence of the present invention is not limited by the above examples.

Preferably, the neural-specific PSEN1 (PS1) expression vector may be the vector disclosed in FIG. 1 .

Therefore, in another aspect of the present invention, it relates to a recombinant vector for neuron-specific expression of PS1 in which the SYN promoter is operably linked to the PS1 gene. Or, in another aspect of the present invention, it relates to a recombinant vector for inserting the SYN promoter and the PS1 gene into the genome of a target, that is, a cell.

The neuron-specific PSEN1 (PS1) expression vector can be used for transforming mutations in which a nucleic acid encoding the PS1 gene is knocked into the genome of a cell. At this time, the knock-in is specific It means that the foreign gene is introduced into the genome of the host so that it can be expressed. A somatic cell into which the recombinant vector has been introduced can be nuclear-transferred into a fertilized egg of an animal, and the nuclear-transferred fertilized egg can be implanted to produce a transgenic individual in which the PS1 gene is knocked in.

The nucleic acid encoding the PS1 gene can be appropriately used by those skilled in the art from sequences having nucleotide sequences encoding the PS1 gene, which are known in the art, but not limited thereto.

A nucleic acid encoding the PS1 gene can be prepared by genetic recombination methods known in the art. For example, PCR amplification for amplifying nucleic acids from a genome, chemical synthesis, or techniques for preparing cDNA sequences, and the like.

In addition, the nucleic acid may have a nucleotide sequence encoding each functional equivalent of PS1.

The functional equivalent refers to a PS1 of the present invention having at least 70%, preferably 80%, more preferably 90% or more sequence homology with the wildtype amino acid sequence as a result of amino acid addition, substitution or deletion. Refers to a polypeptide that exhibits physiological activity substantially equivalent to At this time, deletion or substitution of amino acids is preferably located in a region not directly related to the physiological activity of the polypeptide of the present invention.

Production of transgenic somatic cell lines

Another aspect of the disclosure disclosed herein relates to a method for introducing the neural-specific PSEN1 (PS1) expression vector into a nuclear donor cell and a transgenic somatic cell line into which the neural-specific PSEN1 (PS1) expression vector is introduced. will be.

The transformed somatic cell line may be produced by introducing the neural-specific PSEN1 (PS1) expression vector into a nuclear donor cell.

In this case, the nuclear donor cells may be canine embryonic cells, somatic cells, or stem cells.

In certain embodiments, the nuclear donor cells include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, These include muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal-derived cells, placental cells and embryonic cells. In addition, adult stem cells derived from various tissues of origin, for example, tissue-derived stem cells such as fat, uterus, bone marrow, muscle, placenta, umbilical cord blood or skin (epithelium) may be used. Non-human host embryos can generally be embryos comprising the 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, morula, or blastocyst. .

More preferably, the nuclear donor cells may be fetal-derived cells, adult fibroblasts, or adipose stem cells. Most preferably, canine adipose stem cells are used. The characteristics of these cells are that a large number of cells can be obtained during initial separation, cell culture is relatively easy, and culture and manipulation in vitro are easy.

The embryonic cells, somatic cells, or stem cells provided as nuclear donor cells can be obtained from a method for preparing a surgical specimen or a biopsy specimen using a conventional method known in the art.

The neural-specific PSEN1 (PS1) expression vector can be introduced into the nuclear donor cell by a method known in the art.

For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran - DEAEDextran- mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells The method can be introduced into cells for producing transgenic animals.

Meanwhile, nuclear donor cells transformed with a recombinant vector containing a SYN promoter and a PS1 gene for the production of a neuron-specific PS1 expressing transgenic dog can be propagated and cultured according to methods known in the art.

Suitable media are either developed for the culture of animal cells and in particular mammalian cells, or any use that can be prepared in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and/or micronutrients. Any available medium can be used.

The medium is any basal medium suitable for animal cell growth, as non-limiting examples, basal medium generally used for culture includes MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium) and K-SFM (Keratinocyte Serum Free Medium), and other media used in the industry may be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM/F12 medium, PCM medium, M199/F12 (mixture) (GIBCO), and MSC expansion medium (Chemicon).

To this basal medium can be added anabolic sources of carbon, nitrogen and micronutrients, such as but not limited to serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and/or sugar sources.

It will be apparent that those skilled in the art can properly culture by a known method by selecting or combining suitable media. In addition, it is obvious that culture can be performed while adjusting conditions such as a suitable culture environment, time, and temperature based on conventional knowledge in the field.

[Transgenic animals]

One aspect disclosed by the present specification is to transplant the nuclei of the transformed somatic cell line prepared above, that is, the transformed nuclear donor cell, into enucleated oocytes to produce live offspring, thereby producing neural tissue or cells constituting neural tissue. It relates to a method for preparing a nerve-specific PS1-expressing transgenic dog that specifically expresses PS1 protein in and a nerve-specific PS1-expressing transgenic dog prepared thereby.

In certain embodiments, embryos can be impregnated under suitable conditions to produce dogs that overexpress the PS1 protein specifically in neural tissue or cells constituting neural tissue.

For example, the above somatic cell nuclear transfer (SCNT) method can be used using somatic cells of a transformed dog.

In an arbitrary embodiment, a transgenic animal expressing neuron-specific PS` is produced by somatic cell nuclear transfer (SCNT) using a transgenic cell line in which the PS1 gene is knocked in.

In one embodiment, the method for producing a neuron-specific PS1 expression transgenic dog,

(a) preparing nuclear donor cells comprising culturing somatic cells or stem cells isolated from dogs;

(b) introducing a recombinant vector containing a SYN promoter and a PS1 gene into the nuclear donor cell;

(c) removing nuclei from dog eggs to produce enucleated eggs;

(d) microinjecting and fusing the nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); and

(f) implanting the activated eggs into the oviduct of the surrogate mother.

Conventional descriptions of each step will be understood by referring to methods for producing cloned animals using conventional somatic cell nuclear transfer technology known in the art.

In one embodiment, a method for preparing canine nuclear transfer embryos formed by transplanting nuclei of dog-derived somatic cells, embryonic cells, or stem cells transformed with a composition containing a recombinant vector containing the PS1 gene into enucleated eggs, and the manufacturing method Nuclear transfer embryos can be provided.

In another embodiment, a method for preparing a neuron-specific PS1-expressing transgenic dog in which the PS1 gene is knocked down, comprising the step of transplanting the nuclear transfer embryo into the fallopian tube of a surrogate mother to produce offspring, and characterized in that the PS1 gene produced thereby is knocked in Provided is a transgenic dog expressing neuron-specific PS1.

In certain embodiments, the present invention includes a method for producing a genetically engineered animal,

The method comprises exposing the embryo or cell to a recombinant vector (eg, a recombinant vector containing the PS1 gene),

Cloning cells in the surrogate mother or transplanting embryos into the surrogate mother, wherein the targeting nuclease specifically binds to a target chromosome site in the embryo or cell to cause changes in the cell chromosome, and the surrogate mother causes a change in the target chromosome site Genetically engineered animals can be impregnated.

In one embodiment of the present invention, it was confirmed that the insertion of the nucleic acid sequence encoding the PS1 gene occurred through gene and protein expression analysis of transgenic dogs in which the PS1 gene was knocked in in the present invention.

Using the cells of the transgenic dog in which the PS1 gene has been knocked in, dogs for a neural-specific PS1 expression model in which the PS1 gene is knocked in are produced through re-cloning.

The method of re-replication is not limited, but, for example, the somatic cell nuclear transfer method (SCNT) may be used using somatic cells of a transformed dog.

[purpose]

One aspect disclosed by the present specification provides the use of a transgenic dog in which the PS1 gene is knocked in and the PS1 protein is expressed in a neuron-specific manner.

PS1 is a factor related to Alzheimer's disease (AD) together with Amyloid precursor protein (APP).

Alzheimer's disease (AD) is a representative degenerative cranial nerve disease of cognitive impairment. (1) Neurotoxic substances such as amyloid, an abnormally aggregated protein mass, accumulate in the temporal lobes on both sides, and begin with deterioration of brain function, and gradually The disease progresses as the accumulation of amyloid increases inside and outside the neurons in the cortical part of the brain.

For research and development of therapeutic agents for Alzheimer's disease, disease model animals are also attracting great interest. AD animal models that are currently used in most studies are mouse models. Mice are widely used because they have the advantages of a short breeding cycle, rapid generation change, and easy handling. However, due to breeding and physiological differences with humans, models representing only partial lesions among AD-related lesions are being produced and used.

In some studies, it was confirmed that inducing mutations in APP, PS1 and PS2 using AD mouse models increased the production of beta-amyloid and induced Alzheimer's disease. In addition, it was confirmed that plaque formation similar to Alzheimer's disease was induced in the AD mouse model. In particular, beta-amyloid was exclusively produced and accumulated in APP/PS1 transgenics with 5 FAD mutations, and amyloid accumulation started at 2 months of age and was excessively accumulated in the deep cortical layer and subiculum. In addition, it was observed that neuronal cells were lost due to overexpression of APP/PS1 in APP/PS1 transgenic individuals. When the APP/PS1 transgenic individuals reached 9 months of age, selective loss of nerves was observed, and synaptic junctions were reduced. However, the APP/PS1 transgenic individuals did not show age-dependent cognitive deficit symptoms. It is speculated that the age-dependent loss of cognitive abilities is dependent on the formation of beta-amyloid.

In another study, it was confirmed that the onset of amyloid beta precipitation was earlier in offspring produced by crossing a human carrying the FAD M146V mutation with a rat carrying the PS1 transgene. In addition, the surrounding glial cells and phosphorylated tau lesions were also observed in the lesion pattern, and the amount of beta-amyloid deposits was measured using ELISA, which was similar to that of the mouse model. However, compared to the mouse model, the rat model does not completely match the onset time of each individual, and has the disadvantage that all individuals do not show the same onset progression.

In order to solve the problem of existing disease animal models showing differences between individuals and partial lesions, great efforts have been made to create genetic animal models with similar etiology to Alzheimer's disease, and many transgenic individuals have been created to cause the disease. and development of potential therapeutic agents.

The transgenic dog in which the neuron-specific PS1 protein of the present invention is expressed can be an alternative to a new AD model. Instead of microinjection, which has disadvantages such as low production efficiency and mosaicism, as well as low delivery efficiency in germ cells, it uses a somatic cell nuclear transfer technique without mosaicism, and gene transfer to germ cells is 100% reversed, reducing differences between individuals. , and the human PS1 mutant gene was overexpressed using a nerve-specific promoter to have lesions similar to those of Alzheimer's disease. In addition, transgenic dogs are easy to breed, have a deep bond with people, and are a model that can truly observe the treatment and results of disease models with the development of clinical veterinary medicine.

Therefore, dogs characterized in that the PS1 gene of the present invention is knocked in and the knocked-in PS1 gene is specifically expressed as a protein in nerves have the advantage of easy remodeling, and the nerve-specific PS1 protein ( For example, it is useful as an animal model that overexpresses PS1 mutant protein) and thus has lesions similar to Alzheimer's disease.

That is, the neuron-specific PS1 expression transgenic dog of the present invention will be able to be used in various ways, such as research on mechanisms involved in inducing or treating Alzheimer's disease, the role of PS1, research on the development of therapeutic agents, and development of diagnostic biomarkers.

For example, the animal model according to the present invention can be used as a method for screening drugs for the treatment of Alzheimer's disease.

As one embodiment, the screening method of the present invention

1) administering a candidate substance capable of inhibiting or inhibiting the production of beta amyloid to a transgenic dog expressing neuron-specific PS1;

2) After administering the candidate substance, the dog's neural tissue may be compared with a control group not administered with the candidate substance and analyzed.

Candidate substances are preferably any one selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and blood plasma, but are not limited thereto. The compound may be a novel compound or a widely known compound. These candidate substances may form salts.

As a method of administering the candidate substance as described above, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, intraperitoneal administration, etc. can be appropriately selected depending on the symptoms of the target animal and the properties of the candidate substance. In addition, the dosage of the candidate substance can be appropriately selected according to the administration method or properties of the candidate substance.

For example, the animal model according to the present invention can be used in research related to Alzheimer's disease, for example, as a method of screening for a substance capable of inhibiting or suppressing beta-amyloid plaque formation.

As one embodiment, the screening method of the present invention

1) administering a candidate substance capable of inhibiting or inhibiting the formation of beta-amyloid plaques to a transgenic dog expressing neural-specific PS1;

2) After administration of the candidate substance, it may include the step of analyzing the tissue of the dog by comparing it with a control group not administered with the candidate substance.

Candidate substances are preferably any one selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and blood plasma, but are not limited thereto. The compound may be a novel compound or a widely known compound. These candidate substances may form salts.

As a method of administering the candidate substance as described above, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, intraperitoneal administration, etc. can be appropriately selected depending on the symptoms of the target animal and the properties of the candidate substance. In addition, the dosage of the candidate substance can be appropriately selected according to the administration method or properties of the candidate substance.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

*

Example 1. Establishment of fetal cell line and adipose stem cell line

*For the preparation of nuclear donor cells, canine fetal cell lines and adipose stem cell lines were established.

1. Preparation of Beagle Fetal Cell Line

To establish a fetal beagle cell line, fetuses were recovered from beagles on the 28th day after artificial insemination. After washing the recovered fetus 3 times in PBS, removing the head and organs using fine scissors, cutting the tissue into small pieces, centrifuging using DMEM with 10% (v/v) FBS added, and removing the supernatant. After collecting the tissue and placing it in a culture dish, it is cultured for 6 to 8 days in DMEM supplemented with 10% (v/v) FBS. After culturing, unattached cells or explant masses were removed, and then the attached cells were continuously cultured until confluency to establish a fetal cell line. The established cell lines were frozen using FBS supplemented with 10% DMSO and stored in liquid nitrogen until used in experiments.

2. Preparation of adipose stem cell line derived from beagle adipose tissue

To isolate beagle adipose stem cells, adipose tissue was recovered from the inguinal region of beagle and washed with phosphate buffered saline (PBS). Then, finely chop the tissue using fine scissors, treat with 1 mg/ml collagenase I, and dissolve the tissue by stirring at 37 °C for 60 minutes. After filtering the disintegrated tissue using a 100㎛ cell strainer and centrifuging to obtain a cell fraction, the supernatant is removed and the cells are collected and placed in an adipose stem cell culture medium (RKCM) at 37°C and 5% CO2. cultivate The next day, after removing non-attached cells or tissue lumps from the culture dish, the attached cells were continuously cultured until confluency. In order to verify that the attached cells are stem cells, cell characteristics were analyzed by flow cytometry using a stem cell-specific expression cell surface marker to verify that the cultured cells were adipose stem cell lines. The cell lines thus established were frozen using FBS supplemented with 10% DMSO and stored in liquid nitrogen until used in experiments.

3. Establishment of fetal cell line and adipose stem cell line

To produce a transgenic cloned beagle, fetal beagle cell lines and adult beagle adipose stem cells were established. Until now, skin-derived adult cells among various tissues have been commonly used to clone dogs, but two cell lines have been established to more efficiently produce transgenic dogs. Since both cell lines are maintained in an undifferentiated state, they are cell lines that are usefully used to produce transgenic models in other mammals. The results are summarized in Table 1 below.

Beagle fetal cells recovered 5 fetuses from one pregnant female beagle, of which 2 lines were female and the remaining 3 lines were male, and their sex was confirmed through PCR. All cells were frozen at passage 0, and the established cells are shown in Table 1 below.

Establishment of beagle fetal cell line

Cell Strain	Pssage	Number	Concentration
K9F2010A Male	0 0	4 7	1×10 6 1×10 6
K9F2010B Female	0 0	5 13	1×10 6 1×10 6
K9F2010C Male	0 0	4 16	1×10 6 1×10 6
K9F2010D Female	0 0	9 4	1×10 6 1×10 6
K9F2010E Female	0 0	8 15	1×10 6 1×10 6

After isolating, culturing, and storing adipose stem cells from the fat of a 2-year-old beagle, it was verified that they were adipose stem cells through flow cytometry. The results are shown in FIG. 2 .

Example 2. Production of neural-specific reporter gene-expressing cloned dogs using a neural-specific synapsin promoter

Synapsin promoter-RFP vector was constructed and transgenic dogs were produced to confirm that the RFP gene can be expressed in a neuron-specific manner using the synapsin promoter for neuron-specific gene expression.

1. Preparation of Neuron-specific RFP Expression Vector

A synapsin promoter-RFP vector for neural-specific gene expression was constructed and evaluated. The synapsin promoter-RFP vector uses a Lentivirus vector to regulate gene expression under the SYN promoter, and also introduces a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence to increase the efficiency of mRNA synthesis and thereby improve protein synthesis. induced. To verify a promoter that is specifically expressed in canine nerves, a vector was created by combining the SYN promoter, RFP gene, and phosphoglycerate kinase (PGK) promoter with lentiviral plasmid. After infecting fetal cell lines and adipose stem cells of dogs with the produced virus, the adipose stem cells were differentiated into neurons, and the neuron-specific expression pattern was verified.

2. Production of nerve-specific RFP-expressing transgenic cell lines

A lentiviral vector expressing the RFP gene specifically in canine nerves was constructed using the Human SYN promoter. Beagle adipose stem cells were infected with the constructed viral vector and selected for 4 days using a medium supplemented with 1.5 μg/ml of puromycin. Some of the selected cells were cultured in a neural cell differentiation medium to induce differentiation into neurogenic cells, and the rest were stored frozen.

When the selected cells that had not differentiated into neurons were observed under UV light, RFP was still not observed, but RFP began to be observed in cells differentiated into neurons. Through the expression of RFP in cells on day 14 of neural differentiation, it was verified that the SYN promoter selected for AD model production has the ability to regulate expression of marker genes specifically in neurons in dogs.

3. Production of transgenic dogs expressing neuron-specific RFP

Adipose stem cells expressing SYN-RFP previously established were used to produce neural-specific RFP gene-expressing cloned dogs.

For somatic cell nuclear transfer, SYN-RFP-expressing adipose stem cells were prepared by inducing them into single cells by treating them with trypsin immediately before somatic cell nuclear transfer.

In order to recover mature eggs in the body, blood was collected from the female bitches in the early stages of estrus every day from the lateral cortical vein, and serum was separated and sent to a testing institution to measure blood progesterone levels. After determining the timing of ovulation based on the measured progesterone concentration, mature oocytes were retrieved from the fallopian tubes surgically after 3 days after ovulation under general anesthesia with isoflurane. The recovered internally matured oocytes were transferred to the laboratory within 5 minutes in Hepes-buffered TCM-199 at 38.5°C.

By immersing in 0.1% (v/v) hyaluronidase in Hepes-buffered TCM-199 and pipetting repeatedly, cumulus cells were removed from mature oocytes in vivo and prepared for somatic cell cloning.

Each oocyte was enucleated and each prepared SYN-RFP cell was injected. Then, for the fusion of the cytoplasm of the oocyte and the injected cells, it was placed in a fusion medium containing 0.26 M mannitol, 0.1 mM MgSO4, 0.5 mM Hepes, and 0.05% (w/v) BSA, and 72V, 15 usec, 2 pulses of electricity fusion through stimulation. The fused cloned fertilized egg was treated with 10 μM calcium ionophore for 4 minutes for activation and then cultured in 1.9 mM 6-dimethylaminopurine for 2 hours.

After egg activation, somatic cloned embryos were surgically implanted into the fallopian tubes of surrogate mothers. Surrogate mothers receiving somatic cloned embryos were synchronized with natural estrus. Pregnancy was diagnosed using an ultrasound scanner 26 days after the cloned fertilized egg transfer. Surrogate mothers whose pregnancy was confirmed produced live offspring through caesarean section 60 days after fertilized egg transfer. Produced individuals were evaluated for insertion of foreign genes through genetic analysis.

A total of 82 transgenic cloned fertilized eggs were transferred to a total of 5 surrogate mothers, of which 3 surrogate mothers became pregnant. The pregnant surrogate mothers produced 1 offspring each and successfully produced a total of 3 SYN-RFP cloned dogs. Unfortunately, SYN-R1 died 3 days after birth, and SYN-R2 died shortly after delivery. SYN-R3 is alive and well.

A southern blot experiment was performed to confirm the stable insertion of the foreign gene in the produced SYN-RFP entities. It was confirmed that the gene was inserted in all three offspring, and it was verified that 5 copies were inserted in SYN-R1, 2 copies in SYN-R2, and 1 copy in R3. In addition, when neurons isolated from the brains of deceased SYN-R1 were cultured, RFP was not expressed until the 7th day of culture, but as shown in the figure below, it was observed that the expression of RFP gradually increased from the 8th to the 14th day of culture. could When RFP was observed, it was confirmed that neurons were forming neural spheres.

Through these results, it was confirmed that canine nerve-specific expression is possible using the synapsin promoter, and it is expected that the PS1 gene can be nerve-specifically expressed using the synapsin promoter.

Example 3. Production of nerve-specific PS1 gene expression cloned dog using nerve-specific synapsin promoter

A recombinant vector expressing the PS1 gene in a neuron-specific manner using a synapsin promoter for neuron-specific gene expression and a transgenic dog using the same were produced.

1. Preparation of neuron-specific PS1 expression vectors

Since the PS1 mutant, PS1M146V, was not commercially available, gene synthesis was attempted. The entire PS1 gene base sequence was identified through PubMed's GenBank, and it was confirmed that exactly the 146th amino acid was methionine, and gene synthesis was performed by changing one base sequence so that this amino acid could be converted to valine (FIG. 3).

Under the control of the SYN promoter, the PS1M146V gene is expressed only in nervous tissue, and GFP and Puromycin resistance genes (PuroR) are selected by the PGK (phosphoglycerate kinase) promoter, a systemic expression promoter, to select cells into which the vector has been introduced during somatic cell nuclear transfer. A vector was designed and constructed to express .

2. Production of nerve-specific PS1-expressing transgenic cell lines

After introducing the vector prepared in 1 above into canine adipose stem cells, a cell line expressing SYN promoter-PS1M146V was established through puromycin selection, and the gene insertion was verified at the cell level.

3. Production of transgenic cloned embryos

1) Monitoring of in vitro development of cloned embryos expressing SYN promoter-PS1M146V using interspecies somatic cell nuclear transfer (iSCNT)

In order to evaluate the applicability of somatic cell cloning to somatic cells into which the SYN promoter-PS1M146VP gene was introduced, we first cloned embryos expressing SYN promoter-PS1M146VP using a cross-species somatic cell cloning technique, and observed their development in vitro. The reason why the heterologous somatic cell cloning technique was first performed was that in the case of dogs, due to the characteristics of reproductive physiology, it is difficult to obtain enough mature eggs in the body. Therefore, in this study, the heterologous somatic cell cloning technique using pig eggs was used as a control, a non-transformed cell line, and a SYN promoter. -PS1M146VP transfected cell line was used for comparison. After somatic cell nuclear transfer, the expression of the PSEN1 gene was determined through the expression of the marker gene, GFP.

For interspecies nuclear transfer, porcine oocytes matured in vitro were prepared. The immature oocytes recovered from the ovaries recovered from the slaughterhouse were cultured in an in vitro maturation medium for 40 hours, and then, after removing the cumulus cells, only the oocytes with the first polar body confirmed using a fluorescence inverted microscope were selected using a micromanipulation device. Enucleation was performed. After enucleation, the prepared two types of somatic cells were transplanted into the gastrointestinal tract, respectively, and fusion was performed by electrical stimulation under the condition of 1 DC pulses of 200V/cm, 30 μsec. After 30 minutes of electrical stimulation, somatic cloned fertilized eggs in which somatic cells and egg cytoplasm were fused were selected. Activation of the fused fertilized eggs was induced through electrical stimulation under the condition of 1 DC pulses of 1.5kV/cm, 60 μsec min. Thereafter, the activated cloned fertilized eggs were cultured in vitro for 8 days.

2) Monitoring the in vitro development of cloned eggs expressing SYN promoter-PS1M146V using individual cell cloning technique

2-1) Acquisition of mature eggs in the body

A bitch in the early stage of heat was purchased, blood was collected from the lateral cortical vein every day, serum was separated, and then sent to a testing institution to measure blood progesterone concentration. After determining the timing of ovulation based on the measured progesterone concentration, 3 days after ovulation, general anesthesia with isoflurane was performed, and mature oocytes were retrieved from the fallopian tubes by laparotomy. The first end of the fallopian tube was accessed through the bursal slit and an inverted flanged bulb needle was intubated. (The needle was fixed by surgical ligation, performed using a quick-release device using a 3 cm plastic tube and hemostatic forceps. Blanch the surrounding tissue and lumen of the fallopian tube. Hepes-buffered tissue culture supplemented with 10% (v/v) FBS, 2 mM NaHCO3, 5 mg/ml BSA (Invitrogen, Carlsbad, Calif.) A fine hypodermic syringe (24 gauge) filled with embryo collection medium consisting of Medium (TCM)-199 was intubated. In vivo matured oocytes harvested from flushing were stored in Hepes-buffered TCM-199 at 38.5°C within 10 minutes in the laboratory. moved to

2-2) Preparation of Nucleated Oocytes for Somatic Cell Nuclear Transfer

After being immersed in 0.1% (v/v) hyaluronidase in Hepes-buffered TCM-199 medium, repeated pipetting using a fine glass pipette was performed to obtain an oocyte matured in vivo. Cumulus cells were removed. Then, each oocyte was fixed with a holding micropipette (inner diameter 150 m), and 10% (v/v) FBS and 5 ug/mL bisbenzimide (Hoechst 33342) and 5 ug/mL cytochalasin were added. Enucleation was performed with a micromanipulator (Nikon-Narishige, Tokyo, Japan) in Hepes-buffered TCM-199 medium supplemented with B. Polar bodies and metaphase-II chromosomes stained with bizbenzimide were removed using an aspiration pipette. Enucleated oocytes were placed in TCM-199 supplemented with 10% (v/v) FBS and subsequently used for SCNT.

2-3) Evaluation of microinjection, fusion, activation and fertilized egg culture and in vitro development rate

Enucleated oocytes treated with 100 ug/mL phytohemagglutinin in Hepes-buffered TCM-199 medium supplemented with 10% (v/v) FBS for enhancement of the union of donor somatic cells and nuclear cytoplasm. Single fibroblasts derived from dogs or wolves were injected into the perivitelline space. The couplets were precipitated in a fusion medium containing 0.26 M mannitol, 0.1 mM MgSO4, 0.5 mM Hepes and 0.05% (w/v) BSA, and fused using a needle-type electrode. The single cell-egg complex was placed between two opposing electrodes attached to a micromanipulator (Nikon-Narishige, Tokyo, Japan). The contact surface between the egg cytoplasmic body and the nuclear donor cell was placed parallel to the electrode, and electrical stimulation was applied using an Electro-Cell Fusion apparatus (NEPA GENE Co., Chiba, Japan). The distance between the electrodes is about 180 um (diameter of an egg). 2 pulses were applied at 70-75V, 15 usec duration, and 30 minutes after electrical stimulation, fusion of the donor cell and oocyte cytoplasm was observed under a stereomicroscope. Only fused embryos were selected and cultured for 2 hours in TCM-199 medium supplemented with modified 10% (v/v) FBS as described. The chemical activity of reconstituted dog fertilized eggs was determined by culturing in tubal fluid synthetic medium (mSOF; osmotic pressure and pH were 270 to 280 mOsm and 7.2 to 7.3, respectively) containing 10 µM calcium ionophore at 39°C. Activation of cloned embryos was induced. Thereafter, the replicated fertilized eggs were washed and further incubated for 4 hours in mSOF supplemented with 1.9 mM 6-dimethylaminopurine. After activation of the cloned fertilized egg, it was cultured in vitro for 4 days in the fertilized egg culture medium, and the developmental pattern according to the in vitro culture was observed. After performing somatic cell cloning, the in vitro development rate was observed while culturing for 4 days in tubal fluid synthesis medium in an incubator.

3) Results of monitoring in vitro development of cloned eggs expressing SYN promoter-PS1M146V using interspecies somatic cell cloning technique (interspecies SCNT; iSCNT)

The results of heterologous somatic cell cloning fertilized egg production using somatic cells of dogs and eggs of pigs into which the SYN promoter-PS1M146VP gene was introduced are as follows. In order to verify whether the introduction of the gene affects the development of fertilized eggs of heterologous somatic cell cloning, two groups of somatic cell cloning were performed. The fusion rates of normal cloned embryo groups (Non-transgenic iSCNT) using somatic cells of normal dogs in which no gene was introduced and transgenic cloned embryo groups (Transgenic iSCNT) using transformed cells were respectively, non-transgenic iSCNT embryos, 90% vs. . In transgenic iSCNT embryos, 80% showed a difference. After 24 hours of in vitro culture, the split ratio was 84.2% vs. 88.8%, and there was a difference between the two groups in the proportion of fertilized eggs developed over the 4-cell stage (78.8% vs. 19.6%). In addition, non-transgenic iSCNT embryos, 30.6% vs. blastocyst formation stage in both groups. Differences were shown in transgenic iSCNT embryos, 0.7% (Table 2).

In vitro development of cloned embryos expressing SYN promoter-PS1M146V using heterologous somatic cell nuclear transfer technique

group	Total	2-cell stage (%)	4-cell stage(%)	Blastocyst stage (%)
Non-transgenic iSCNTs	101	85 (84.2)	67 (78.8) b	26 (30.6) b
Transgenic iSCNTs	322	286 (88.8)	56 (19.6) a	2 (0.7) a

This difference is interpreted as a result of inefficient reprogramming after fusion of donor cells into which the SYN promoter-PS1M146VP gene was introduced through genetic manipulation of cells used for somatic cell nuclear transfer. In addition, as shown in the figure below, embryos derived from donor cells transfected with the SYN promoter-PS1M146VP gene expressed GFP in 2-cell, 4-cell, and blastocysts, but did not show strong expression, but mosaicism at all stages not observed

4) Monitoring results of in vitro development of cloned eggs expressing SYN promoter-PS1M146V using individual cell cloning technique

In vitro development of dog cloned embryos was observed using dog somatic cells and dog eggs into which the SYN promoter-PS1M146VP gene was introduced.

After performing individual somatic cell cloning, the in vitro development rate was observed while culturing in vitro for 4 days. As shown in the table below, the fusion rate after somatic cell cloning was 82.1%. After 24 hours of in vitro culture, the division rate was 50%, the proportion of embryos reaching the 4-cell stage was 46.9%, and the 8-cell stage showed a development rate of 28.1%. However, the current in vitro culture system of dogs has not been established, so further development could not be observed during in vitro culture.

In addition, it was confirmed that the expression of the maker gene, GFP, was expressed in all fertilized eggs into which the SYN promoter-PS1M146V gene was introduced. Therefore, the results so far confirmed the basis for producing SYN promoter-PS1M146V-expressing clones using SYN promoter-PS1M146V donor cells.

In vitro development of cloned eggs expressing SYN promoter-PS1M146V using canine cell cloning technique

Replication	TotalNo. oocyte	No. of fused oocytes (%)	No. 2-cell stage (%)	No. 4-cell stage (%)	No. 8-cell stage (%)
A	8	7(87.5)	4(57.1)	4(57.1)	3(42.9)
B	12	9(75.0)	4(44.4)	3(33.3)	1(11.1)
C	19	16(84.2)	9 (56.3)	8(50.0)	5 (31.3)
D	6	2(33.3)	1(50.0)	1(50.0)	0(0)
Total	45	34 (75.5)	18(40.0)	16 (35.5)	9(20.1)

4. Production of transgenic dogs expressing neuron-specific PS1

1) Transfer of cloned fertilized eggs to a surrogate mother for production of transgenic cloned dog and production of transgenic cloned dog

Dog somatic cells into which the SYN promoter-PS1M146VP gene was introduced were used.

The change in serum progesterone concentration predicts the ovulation day of the egg donor dog, and mature eggs are recovered from the fallopian tube of the egg donor dog. After removing cumulus cells from hyaluronidase of mature oocytes, oocytes with a first polar body and a gastrocranial space of 15-25 μm in width were selected and used for somatic cell nuclear transfer.

The nucleus of the selected oocyte is removed to prepare an enucleated oocyte, and a cell expressing the SYN promoter-PS1M146VP gene is injected into the enucleated oocyte, using the Electro-Cell Fusion apparatus (NEPA GENE, Chiba, Japan) for 2 pulses Fusion between oocyte cytoplasm and cells was induced by electric stimulation of direct current (72 V for 15 ms). The fused egg-cell fusions were selected, and fusion between the donor cell and the ovum cytoplasm was observed under a stereomicroscope 30 minutes after electrical stimulation. Only fused embryos are selected and cultured for 2 hours in TCM-199 medium supplemented with 10% (v/v) FBS modified as described. The chemical activity of reconstituted dog fertilized eggs was measured in tubal fluid synthetic medium (mSOF; osmotic pressure and pH were 270 to 280 mOsm and 7.2 to 7.3, respectively) containing 10 µM calcium ionophore at 39°C for 4 minutes. After incubation, cloned fertilized eggs were washed and further incubated for 4 hours in mSOF supplemented with 1.9 mM 6-dimethylaminopurine.

After activation of the cloned embryo was completed, the cloned embryo was loaded and transplanted into the fallopian tube of the surrogate mother synchronized with natural ovulation using a Tamcat catheter. After intrafallopian transplantation of the estrus-synchronized surrogate mother, pregnancy was diagnosed by screening the surrogate mother's abdomen with ultrasound 26 days after the date of transplantation, and the number of fetuses was confirmed using X-ray after 45 days of confirmed pregnancy. After collecting blood once or twice a day from around 52 days, the trend of P4 decrease was analyzed and the fetal heart rate was observed by ultrasound at the same time. If it decreased to , a cesarean section was performed.

In vivo development of cloned eggs expressing SYN promoter-PS1M146V using canine cell cloning technique

Recipient	Transferred embryos	Pregnancy	Size of litter	Puppy ID
A	13
B	8	-
C	12	+	One	PS1
D	6	-
E	7	-
F	8	-
G	12	-
H	10	-
I	12	-
J	7	-
K	10	+	One	PS1-1*
L	9	-
M	8	-
Total	122	15.4%	1.6%

* Died of pneumonia on the 19th day after birth

2) Production of PS1M146VP gene expression transgenic cloned dog

122 cloned fertilized eggs were transplanted into 13 surrogate mothers using PS1M146VP gene-expressing donor cells, and as a result of ultrasound pregnancy diagnosis about 26 days after transplantation, it was confirmed that 2 surrogate mothers were pregnant. On the 52nd day after transplantation, an X-ray was taken to confirm the number of live births. In addition, two transgenic cloned dogs were successfully produced from two surrogate mothers through caesarean section on the 60th day of pregnancy. However, around 15 days after birth, one of the two transgenic cloned dogs produced died of pneumonia, and subsequent evaluation was conducted only through one living individual.

3) Verification of genotype and expression of PS1M146VP gene expression transgenic cloned dogs

In order to verify whether the PS1M146VP gene was inserted into the genome, it was analyzed using general beagle blood of the same age (negative control), cloned individuals, and plasmid vector (positive control). The primers used in the analysis are summarized in FIG. 4 . As shown in FIG. 4, it was confirmed that the hSyn1p-PS1 gene was expressed in the transgenic cloned dogs.

The results of verifying the insertion of the transferred foreign gene by Southern blotting are shown in FIG. 5 . In FIG. 5, A) of the transgenic clone PS1 was subjected to 2 cuts with restriction enzyme to verify insertion of the PS1M146VP gene, and B) was performed with 1 cut using HindIII enzyme to confirm that a total of 10 copies or more were inserted.

Physiological normality was evaluated when the transgenic cloned dog reached the age of 6 months, and it was confirmed that all hematological and serological values were within the normal range. It was confirmed that he was physically healthy until now, when he was 6 months old.

5. Analysis of physiological changes in biomarkers of cognitive impairment in PSEN1-expressing dogs (beagles)

Changes in canine APP (beta-amyloid precursor protein), canine Tau, human PSEN-1, and canine PSEN-1 were observed through ELISA analysis using serum from PSEN1-expressing beagles, beagles of the same age, and older dogs with cognitive impairment. The detailed method is as follows.

1) Experiment method

For blood collection of beagles, after depilation around the blood collection site with clippers, disinfection with 70% alcohol or chlorhexidine, about 8ml of blood is collected from the jugular vein or the radiolateral cutaneous vein, and 1.5ml and 5ml each in 2 SST tubes and 0.5ml in EDTA tube was inserted into the whole blood cell analyzer, and CBC (Complete Blood cell Count), differential counting, and serum chemistry analysis were performed.

Analysis items are WBC (White blood cell) count, RBC (Red blood cell) count, Hb (Hemoglobin) concentration, PCV (Packed cell volume, hematocrit), MCV (Mean corpuscular volume), MCH (Mean corpuscular hemoglobin) , MCHC (Mean corpuscular hemoglobin concentration), RDW (Red cell distribution width), and platelet count (PLT) were examined. After confirming that the blood collected in the SST tube was coagulated, the serum was separated by centrifugation at 1500 rpm for 5-10 minutes, and the serum in the upper layer was reacted with the sample to perform quantitative analysis, Serum chemistry and ELISA analysis. The analysis items included alkaline phosphatase (ALP), calcium, phosphorus, and magnesium, and were performed to analyze the normality of PSEN1-expressing beagle.

After ELISA analysis, the enzyme-linked immunosorbent assay (ELISA) kit (LifeSpan BioSciences Inc, Seattle, WA , U.S.A.) and the detailed method is as follows:

a) Before starting ELISA, all reagents were kept at room temperature and mixed homogeneously by pipetting.

b) Put 100ul of standard, blank and sample, cover with sealer, and incubate for 1 hour at 37 degrees,

c) Discard all the liquid in each well, and do not wash at this time,

d) After adding 100ul of detection reagent A and covering it with a sealer, tap the plate lightly to mix the solution homogeneously, and incubate at 37 degrees for 1 hour,

e) After discarding the liquid in each well, wash 3 times using wash buffer (after adding washing buffer for each washing process, leave it for about 1-2 minutes),

f) After the last washing, invert the plate on a clean paper to completely absorb the liquid before removing it.

g) After removing the wash buffer, add 100ul of Detection Reagent B to each well, cover it with a plate sealer, and incubate at 37 degrees for 30 minutes,

h) After discarding the liquid in each well, wash a total of 5 times using wash buffer,

i) After the last washing, the wash buffer was removed, and 90ul of TMB substrate was added to each well,

j) Cover with a plate sealer and incubate in a shaded environment at 37 degrees for 10-20 minutes, and

k) After adding the stop solution to each well, tap the plate lightly and measure the absorbance at 450 nm using a microplate reader.

2) Blood/serum analysis results

*The results of confirming the health status of the PSEN1 expressing object through hematological/serological analysis were as follows and confirmed that they were normal (FIG. 6), and then the analysis experiment was conducted.

3) Analysis of changes in three types of canine APP, canine Tau, human PSEN-1, and canine PSEN1 cognitive impairment biomarkers using ELISA

As a result of statistical analysis through a total of 3 repeated experiments using the serum of 1 PSEN1-expressing beagle, 2 elderly dogs, and 3 beagles of the same age who participated in this study, the expression of cognitive disorder biomarkers showed differences according to the experimental groups. .

In the PSEN1 ELISA assay, canine PSEN1 and human PSEN1 concentrations showed different expression patterns. As shown in Figure 7, in the case of canine PSEN1, there was no difference in expression concentration by group. However, in the case of human PSEN1, the PSEN1-expressing beagle showed the highest concentration compared to the other two groups, and the Old group showed a significantly higher concentration than the Young group, but showed a lower concentration than the PSEN1-expressing beagle.

In the results of Canine Tau analysis, the PSEN1 expression beagle was significantly higher than the Young group, but there was no significant difference from the Old group. These results show that human PSEN1 can induce dog Tau expression (FIG. 8).

In the results of Canine APP analysis, all three groups showed low expression rates, and the Old group showed significantly high expression (FIG. 9). Further follow-up is necessary to study the expression mechanism between APP and PSEN1.

6. Analysis of imaging changes using MRI of PSEN1-expressing beagles

Structural changes in the brain were observed using MRI scans of PSEN1-expressing beagles and beagles of the same age. All subjects were examined before MRI and before anesthesia, and a neurological examination was performed after confirming that there was no abnormality.

1) Experiment method

go. MRI scan

Propofol 6mg/kg was injected intravenously, and anesthesia was maintained using isoflurane 1.5-2 MAC. For MRI, images were obtained using a 16-channel knee coil while lying on the stomach. The patient is photographed from the olfactory bulb of the brain to the tip of the cerebellum, the shooting interval is maintained at 3 mm, transverse and sagittal sections are obtained, and the MRI scan includes all of the cerebrum from the beginning to the end, as follows The filming was carried out in the same sequence. (1) T2-weighted image (T2-weighted image), T1-weighted image (T1-weighted image), GRE (Gradient recalled echo). The size of the ventricles was measured on T2-weighted images, and hemorrhagic changes in the cerebral parenchyma were confirmed on the GRE.

me. MRI measurement and analysis

The captured images were converted into DICOM format and measured using Dicom viewer. For measurement, a commercially available DICOM viewer was used, and each item was measured as shown in FIG. 10. Considering the error, all measured values were evaluated as the average value after measuring three times. Statistical analysis was conducted for each of the three groups to confirm significant differences. The height of lateral ventricles (mm), cerebral height (mm), and the ratio of ventricular height to cerebral height are interthalamic adhesion (ITA), the ratio of interthalamic adhesion to cerebral height, and the height of the third ventricle to cerebral parenchymal height. The ratio measured the thickness of the cerebral cortex and the adjacent sulcus in the T2 image. The measurement area was measured between the logitudinal fissure and the first cerebral sulcus, the ectomarginal sulcus. The thickness of the cerebral cortex, the width of the sulcus, and the ratio were measured, and the average value was derived after measuring three times to minimize the error of the measurer. The presence or absence of white matter hyperintensity in the internal capsule, which is the cerebral white matter, was confirmed.

all. Diffusion Tender Imaging (DTI) Evaluation of the Brain of Experimental Dogs

DTI values were calculated using MRI in dogs, anesthesia and MRI imaging were performed under the same conditions, and changes in white matter according to aging were confirmed. DTI imaging is limited to the cerebral hemisphere, and the white matter is divided into a total of 9 areas to be photographed and measured individually, and the 9 selected areas are shown in FIG. 11 . ADC (apparent diffusion coefficient) and FA (fractional anisotropy) values were measured at the corresponding site for each individual, and color-coded DTI and tractography were used to confirm overall white matter connectivity.

2) Experimental results

go. Assessment of the two-dimensional structure of the brain using MRI

For MRI evaluation, imaging was performed on 1 PSEN1-expressing beagle (hereinafter referred to as PSEN-1) and 1 beagle (hereinafter referred to as Ori11) of the same age. As a result, significant differences in cerebral atrophy and cortical sulcus expansion in PSEN1-expressing beagles were not confirmed. Moderate lateral ventricular enlargement (right 20%, left 18% relative to cerebral hemisphere height) was observed in PSEN1-expressing beagles. Interthalamic junction thickness of the PSEN1-expressing beagle was within the normal range (7.63 mm), and leukoaraiosis was not confirmed in the T2 phase of the PSEN1-expressing beagle. In addition, compared to beagles of the same age, it was confirmed that the thickness of the epidural fat of the PSEN1-expressing beagle increased (Figs. 12 to 18).

me. MRI scan results for evaluation of diffusion tensor imaging (DTI) of the brain

For MRI evaluation, imaging was performed on 1 PSEN1-expressing beagle (hereinafter referred to as PSEN-1) and 1 beagle (hereinafter referred to as Ori11) of the same age. As a result, compared to beagles of the same age, the FA values of body corpus callosum, splenium corpus callosum, and fornix of PSEN1-expressing beagles decreased. MD values of hippocampus and fornix of PSEN1-expressing beagles were increased compared to beagles of the same age. In addition, compared to beagles of the same age, atrophy of splenium corpus callosum was confirmed on the color FA map of PSEN1-expressing beagles (FIGS. 19 to 23).

all. A Study on Brain Imaging Analysis Results

In DTI (MD, FA), findings that are thought to be due to the aging process of the PSEN1-expressing beagle were confirmed, but no obvious aging findings were identified in the remaining sequences.

In the case of humans, changes in MD and FA values were confirmed according to age. In the case of corpus callosum, FA increased until the age of 20 and decreased after the age of 20, and MD showed the opposite trend. In addition, atrophy of the corpus callosum on the color FA was confirmed in patients with Alzheimer's disease. Decreased FA values and atrophy on the color FA map of corpus callosum identified in PSEN1-expressing beagles in this test are thought to reflect aging. Increased MD values of the hippocampus and fornix can be considered for increased diffusivity due to thinned myelin sheath and increased extracellular space due to dysmyelination due to axon aging, but continuous follow-up is required.

7. Semen evaluation of PSEN1-expressing beagles and beagles of the same age and verification of freezing and breeding ability

The semen of the beagle was collected and the normality of the semen was evaluated using CASA. Semen that received normal evaluation was frozen three times to preserve germ cells. The collected semen was intended to proliferate PSEN1-expressing beagles by producing progeny through artificial insemination with normal female beagles.

1) Experiment method

When swelling was observed after stimulating the male's bulbous glands using digital friction therapy, the foreskin was quickly pulled to the back of the bulbous glands to induce exposure of the penis and the bulbous glands. At this time, using a disposable artificial vagina, press the thumb and forefinger on the penile gland to achieve sufficient erection, then spontaneous pelvic movement and a small amount of prostatic fluid are ejected, followed by two fractions of semen, which account for most of the sperm. of ejection was collected. While raising the hind limbs, they take a posture of turning backwards. At this time, since the prostatic fluid, which is mainly 3 fractions, is released, it is better to remove it for freezing of semen, so up to 2 fractions were collected.

To analyze quality aspects such as sperm motility and shape, 10 μl of semen was placed in a Makler counting chamber (ZDL, USA), and a stereomicroscope (Olympus, Tokyo, Japan) connected to a CCD camera (Toshiba, Tokyo, Japan) was used. Japan) and evaluated using the sperm image analysis system shown in the figure above using SIAS (SIAS, Medical supply, Seoul, Korea).

The present invention relates to the production of an Alzheimer's disease (AD) model cloned dog using a somatic cell nuclear transfer technique. More specifically, it relates to the development of cells in which the human PS1 mutant gene, one of the human AD pathogenesis genes, is overexpressed, and the production of transgenic dogs expressing PS1 using the same.

Claims (15)

1. A transgenic dog expressing human PSEN1 (presenilin-1),

   The transgenic dog has a genome containing a human PSEN1 gene,

   At this time, the transgenic dog, characterized in that the human PSEN1 gene is a gene encoding a presenilin-1 mutant protein.
2. According to claim 1,

   Transgenic dog, characterized in that the human PSEN1 gene is operably linked to the synapsin (SYN) promoter.
3. According to claim 2,

   Transgenic dog, characterized in that the human PSEN1 gene is specifically expressed in nervous tissue.
4. According to claim 1,

   The transgenic dog, characterized in that the presenilin-1 mutant protein is a mutation related to Alzheimer's disease.
5. According to claim 4,

   The presenilin-1 mutant protein is an I416T mutation, A79V mutation, H163R mutation, F388L mutation, I143T mutation, L381V mutation, C410Y modified mutation, L286V mutation, L435F mutation or M146V mutation Transgenic dog, characterized in that.
6. According to claim 5,

   The transgenic dog, characterized in that the presenilin-1 mutant protein is an M146V mutant.
7. According to claim 1,

   The transgenic dog is a transgenic dog, characterized in that the cognitive disorder animal model.
8. According to claim 7,

   Transgenic dog, characterized in that the cognitive disorder is Alzheimer's disease (AD).
9. (a) preparing nuclear donor cells comprising culturing somatic cells or stem cells isolated from dogs;

   (b) introducing a recombinant vector containing a SYN promoter and a human PSEN1 gene into the nuclear donor cell;

   (c) removing nuclei from dog eggs to produce enucleated eggs;

   (d) microinjecting and fusing the nuclear donor cells of step (b) into the enucleated oocytes of step (c);

   (e) activating the oocyte fused in step (d); and

   (f) implanting the activated egg into the fallopian tube of the surrogate mother

   Method for producing a transgenic dog expressing human PSEN1 (presenilin-1) comprising a.
10. According to claim 9,

    Wherein the human PSEN1 gene is a gene encoding a presenilin-1 mutant protein.
11. According to claim 10,

    Wherein the presenilin-1 mutant protein is a mutation related to Alzheimer's disease.
12. According to claim 11,

    Wherein the presenilin-1 mutant protein is an I416T mutation, A79V mutation, H163R mutation, F388L mutation, I143T mutation, L381V mutation, C410Y modified mutation, L286V mutation, L435F mutation or M146V mutation.
13. According to claim 12,

    Wherein the presenilin-1 mutant protein is an M146V mutant.
14. According to claim 9,

    The transgenic dog is a transgenic method, characterized in that the cognitive disorder model animal.
15. According to claim 14,

    The method of claim 1, wherein the cognitive disorder is Alzheimer's disease (AD).

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