Expression cassette and vector comprising Alzheimer's disease-related mutant genes and cell line transformed by means of same

Abstract

The present invention relates to expression cassettes and vectors comprising Alzheimer's disease-related genes and a cell line transformed by the disclosed expression cassettes and vectors, and more specifically, the expression cassettes according to the present invention comprise amyloid precursor protein (APP), Tau protein, and presenilin-1 (PS1) genes associated with Alzheimer's disease so that mutant genes thereof can be simultaneously expressed. Additionally, the present invention relates to a cell line transformed by the disclosed expression cassettes or vectors comprising APP, Tau protein, and PS1 genes, and further, to an animal model transformed by the vectors or cell line.

Claims

1. A recombinant expression vector comprising an expression cassette associated with an Alzheimer's disease (AD), the expression cassette comprising (a) a mutant amyloid precursor protein (APP) gene for encoding an APP, (b) a mutant tau gene for encoding a tau protein, (c) a mutant presenilin 1 (PS1) gene for encoding a PS1 and (d) a neuron-specific promoter for controlling the mutant APP gene, the mutant tau gene and the mutant PS1 gene all at once, wherein the mutant APP gene, the mutant tau gene and the mutant PS1 gene are connected in tandem, and, wherein the recombinant expression vector comprises the sequence of SEQ ID NO: 9.

2. A transgenic mammalian cell line comprising the recombinant expression vector of claim 1.

3. A transgenic non-human mammal comprising the recombinant expression vector of claim 1.

4. The transgenic non-human mammal of claim 3, wherein the mammal is a pig.

5. A method of producing the recombinant expression vector of claim 1, the method comprising: (i) constructing a pTet-CKOS derived first vector, the first vector comprising a restriction enzyme site insertion; (ii) inserting a human platelet-derived growth factor (hPDGF)-chain promoter, an amyloid precursor protein (APP) gene, a tau gene, and a presenilin 1 (PS1) gene into a psCMV vector, to obtain a recombinant second vector; (iii) introducing a mutation in each of the APP gene, the tau gene, and the PS1 gene of the recombinant second vector; and (iv) inserting an expression cassette comprising the hPDGF -chain promoter, the mutant APP gene, the mutant tau gene, and the mutant PS1 gene which are connected in tandem, from the recombinant second vector into the first vector to produce the recombinant expression vector comprising the sequence of SEQ ID NO: 9 of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1A is a diagram illustrating an original pTet CKOS retroviral vector.

(2) FIG. 1B is a diagram illustrating a modified pTet vector. FIG. 1B discloses SEQ ID NO: 34.

(3) FIG. 2 is a diagram illustrating a structure of a psCMV vector including a human platelet-derived growth factor (hPDGF) -chain promoter, a human amyloid precursor protein (hAPP) gene, a human tau (hTau) gene and a PSEN1 gene.

(4) FIG. 3A is a diagram illustrating a one-dimensional (1D) structure of a multi-cistronic vector of a pTet retrovirus manufactured so that an hAPP gene, an hTau gene and a PSEN1 gene are expressed using a CMV enhancer and an hPDGF- promoter.

(5) FIG. 3B is a diagram illustrating a cyclic structure of a multi-cistronic vector of a pTet retrovirus manufactured so that an hAPP gene, an hTau gene and a PSEN1 gene are expressed using a CMV enhancer and an hPDGF- promoter.

(6) FIG. 4 is a diagram illustrating primers used to verify integration of a multi-cistronic vector of a pTet retrovirus. FIG. 4 discloses SEQ ID NOS 35-44, respectively, in order of appearance.

(7) FIG. 5 is a photograph illustrating 16 cell lines of a pig based on a multi-cistronic vector of a pTet retrovirus.

(8) FIG. 6 illustrates a result of a deoxyribonucleic acid (DNA) fragment analysis of S0227 among short tandem repeat (STR) sites of each of a surrogate mother, a donor cell and cloned pigs.

(9) FIG. 7 is a table showing a result of a DNA fragment analysis of S0227, S0018 and SW316 among STR sites of each of a surrogate mother, a donor cell and cloned pigs.

BEST MODE FOR CARRYING OUT THE INVENTION

(10) Hereinafter, embodiments will be described in detail with reference to the accompanying drawings.

(11) Embodiments may provide an expression cassette associated with an Alzheimer's disease (AD). The expression cassette may include a) a mutant amyloid precursor protein (APP) gene for encoding an APP, b) a mutant tau gene for encoding a tau protein, c) a mutant presenilin 1 (PS1) gene for encoding a PS1 and d) a neuron-specific promoter for controlling the mutant APP gene, the mutant tau gene and the mutant PS1 gene all at once.

(12) The neuron-specific promoter may include, for example, all promoters enabling a gene to be specifically expressed in neurons, regardless of a type of promoters. For example, the neuron-specific promoter may be a human platelet-derived growth factor (hPDGF) -chain promoter that has been known as a promoter to allow an exotic gene to be expressed in a brain cell of a pig.

(13) The expression cassette may further include an enhancer to further enhance expression of a gene. The enhancer may include, for example, a cytomegalovirus (CMV) enhancer.

(14) The expression cassette may further include 2A sequences between the mutant APP gene, the mutant tau gene and the mutant PS1 gene. For example, a 2A sequence may be inserted between the mutant APP gene and the mutant tau gene, and a 2A sequence may be inserted between the mutant tau gene and the mutant PS1 gene.

(15) A 2A gene sequence according to an embodiment may code 18 to 22 amino acids. Among the amino acids, four amino acids located in a terminal, that is, asparagine (N), proline (P), glycine (G) and proline (P), are importantly preserved between species. The 2A gene sequence tends to self-cleave in synthesis of peptide. Due to the above properties, when a ribosome reaches sites of genetic code encoding N, P, G located in a terminal of a 2A sequence during a protein transcription, the ribosome may sequentially recognize N, P, G and may form peptide bonds. The ribosome may bring a releasing factor (RF) instead of a prolyl-transfer ribonucleic acid (tRNA) on a site in which proline is encoded. When the RF is bonded, a peptide formed in advance may not be connected to the peptide bond any more, and may be discharged from the ribosome. A code encoded after the 2A sequence may operate normally to perform a next protein transcription. As a result, by inserting a 2A sequence, a plurality of genes may be expressed using a single promoter. According to an embodiment, the expression cassette may simultaneously express the above three genes by inserting 2A sequences between the three genes.

(16) The mutant APP gene may have a mutation at amino acid position 595, amino acid position 596, or both, and the mutant tau gene may have a mutation at amino acid position 243. The mutant PS1 gene may have a mutation at amino acid position 146, amino acid position 286, or both. For example, the mutant APP gene may be a gene in which lysine (Lys) is mutated to asparagine (Asn) at amino acid position 595 in APP695 and methionine (Met) is mutated to Lys at amino acid position 596. The mutant tau gene may be a gene in which phenylalanine (Phe) is mutated to Lys at amino acid position 243. The mutant PS1 gene may be a gene in which Met is mutated to leucine (Leu) at amino acid position 146 and proline (Pro) is mutated to Leu at amino acid position 286.

(17) The mutant APP gene may have a sequence of SEQ ID NO: 4 and the mutant tau gene may have a sequence of SEQ ID NO: 5. The mutant PSI gene may have a sequence of SEQ ID NO: 6.

(18) Embodiments may provide a recombinant expression vector including the expression cassette. In the present disclosure, all expression vectors to induce efficient expression of an AD-related protein with specificity to neurons may be used regardless of a type of expression vectors, and a retroviral vector may desirably be used.

(19) Also, embodiments may provide a cell line transformed using the recombinant expression vector, and provide an animal that is other than a human and that is transformed using the cell line.

(20) The cell line may include, for example, cell lines of animals derived from mammals other than a human with a limitation. However, since a commonly used mouse has an extremely high metabolic rate and a change in a life cycle of the mouse is completely different from that of a human, it has been difficult to use the mouse as an accurate disease model. Thus, an animal having a size similar to a body size of a human and similar in metabolism to the human may desirably be used, and most desirably, a pig may be used. When a pig transformed using a mutant gene of an AD produced according to an embodiment is used, there is an advantage in that studies on actions of three genes associated with the AD may be simultaneously conducted by simultaneously creating the three genes. In particular, research on a change in a nervous system as a problem in the AD by neuron-specific expression may be focused.

(21) Hereinafter, the present disclosure will be described in detail with reference to examples. The examples are merely intended for the purpose of describing the present disclosure, and the scope of the present disclosure is not limited or restricted to the examples.

EXAMPLE 1

Construction of New Retroviral Vector

(22) A tetracycline (Tet) responsive element (TRE) minimal CMV promoter and a CKOS gene cluster were eliminated from pTet-CKOS that is a retroviral vector. The retroviral vector was modified to have restriction enzyme sites, for example, BglII, SwaI, PacI, NotI and XhoI, to be advantageous for gene cloning.

(23) FIG. 1A is a diagram illustrating an original pTet CKOS retroviral vector.

(24) FIG. 1B is a diagram illustrating a modified pTet vector.

(25) Referring to FIGS. 1A and 1B, the TRE minimal promoter and a CKOS part (5,568 base pairs (bp)) were eliminated from the original pTet-CKOS vector with a total length of 13,320 bp, and a multiple cloning site of 76 bp was inserted in a site in which the TRE minimal promoter and the CKOS part were eliminated. An example of a base sequence of the pTet vector with a modified structure may be defined with reference to SEQ ID NO: 1 described in the accompanying sequence list.

EXAMPLE 2

Introduction of CMV Enhancer, hPDGF Promoter and AD Gene

(26) A primer including a restriction enzyme site was manufactured and used to insert, into a vector, an hPDGF promoter, a CMV enhancer, and an APP gene (NM_201414.2) of a -amyloid, a PS1 gene (NM_000021.3), a tau gene (NM_016834.4), and the like that cause the AD.

(27) TABLE-US-00001 TABLE1 Target Primername Sequence(5 -> 3) Size pTet-enz. pTet-Enz- AGATCTATTTAAATACCGGT(SEQID 67bp insert insert-F NO:10) pTet-Enz- CTCGAGGCGGCCGCCCTGCA(SEQID insert-R NO:11) hPDGFb BamH1-Swa1- GGATCCATTTAAATGCTGGGACTACAGGA 1,530bp promoter PDGFb-F GCTTG(SEQIDNO:12) PDGFb-Cla1-R ATCGATGTGCGCGCAAAGTATCTCTA (SEQIDNO:13) hAPPswcDNA Cla1- ATCGATATGCTGCCCGGTTTGGCACT 2,106bp hAPPsw-F (SEQIDNO:14) hAPPsw- GGCATGCTTAATTAAGTTCTGCATCTGCT Pac1-Sph1-R CAAAGA(SEQIDNO:15) hTaucDNA Bgl2-hTau-F AGATCTATGGCTGAGCCCCGCCAGGA 1,161bp (SEQIDNO:16) hTau-EcoR1-R GAATTCCAAACCCTGCTTGGCCAGGG (SEQIDNO:17) 2Apeptide EcoR1-2A-F GAATTCGGAAGCGGAGCTACTAACTT 78bp (SEQIDNO:18) 2A-Xhol-R CTCGAGAGGTCCAGGGTTCTCCTCCA (SEQIDNO:19) hPS1cDNA Xho1-hPS1-F CTCGAGATGACAGAGTTACCTGCACC 1,424bp (SEQIDNO:20) hPS1-Xba1-R TCTAGACCTGCAGGCTAGATATAAAATTG ATGGA(SEQIDNO:21) CMV CMVE-F ATTTAAATGCGTTACATAACTTACGG 321bp enhancer (SEQIDNO:22) CMVE-R ATTTAAATCATGGTAATAGCGATGAC (SEQIDNO:23)

(28) Table 1 shows used primers.

(29) The CMV enhancer and PDGF promoter were hybridized and used as a promoter so that AD-related genes are overexpressed with specificity to brain neurons. Before constructing a retroviral vector as a final vector, each mutant gene, the CMV enhancer and PDGF promoter were inserted into a psCMV vector. To insert a gene into a psCMV vector, a polymerase chain reaction (PCR) was used for amplification and cloning was performed using a restriction enzyme site introduced into a vector.

(30) FIG. 2 is a diagram illustrating a structure of a psCMV vector including an hPDGFb promoter, a human APP (hAPP) gene, a human tau (hTau) gene and a PSEN1 gene manufactured according to an embodiment. An example of an SV40 poly A sequence may be defined with reference to SEQ ID NO: 7.

EXAMPLE 3

Mutagenesis of AD Gene

(31) To induce a mutation in an amino acid of an AD related gene after inserting the AD related gene into a psCMV vector, a site-directed mutagenesis kit (Stratagene) was used.

(32) TABLE-US-00002 TABLE2 Target Primername Sequence(5 -> 3) hAPPSw hAPPsw- GAGATCTCTGAAGTGAATCTGGATGCAGAATTCCGA Mutagenesis-1 M1(KM/NL)-F (SEQIDNO:24) hAPPsw- TCGGAATTCTGCATCCAGATTCACTTCAGAGATCTC M1(KM/NL)-R (SEQIDNO:25) hAPPSw hAPPsw- GTCATAGCGACAGTGGTCATCATCACCTTGGTGATG Mutagenesis-2 M2(IV/VI)-F (SEQIDNO:26) hAPPsw- CATCACCAAGGTGATGATGACCACTGTCGCTATGAC M2(IV/VI)-R (SEQIDNO:27) hTau hTau-M(P/L)-F AATATCAAACACGTCCTGGGAGGCGGCAGTGTGC Mutagenesis (SEQIDNO:28) hPS1 hTau-M(P/L)-R CACACTGCCGCCTCCCAGGACGTGTTTGATATT Mutagenesis-1 (SEQIDNO:29) hPS1- AGTGTCATTGTTGTCCTGACTATCCTCCTGGTG M1(M/V)-F (SEQIDNO:30) hPS1- CACCAGGAGGATAGTCAGGACAACAATGACACT M1(M/V)-R (SEQIDNO:31) hPS1 hPS1- TGAAACGCTTTTTCCAGCTGTCATTTACTCCTCAACA Mutagenesis-2 M2(L/V)-F (SEQIDNO:32) hPS1- TGTTGAGGAGTAAATGACAGCTGGAAAAAGCGTTT M2(L/V)-R CA(SEQIDNO:33)

(33) Table 2 shows primers used for mutagenesis.

(34) As an APP gene, an APP695 gene expressed in brain neurons was used, and double mutations were introduced at amino acid positions 595 and 596 in which familial mutations of the AD have been found. The mutations are known to form a larger amount of -amyloid 42. The mutations are called K595N and N596M. In a presenilin gene, two amino acid mutations were introduced. Mutations were introduced in amino acid positions 146 and 286 and are called M146L and P286L, respectively. In a tau gene, a single mutation occurs at amino acid position 243 and is called P243L.

(35) To separate three mutant genes as independent peptides when the three mutant genes are translated to proteins after transcription to a single messenger RNA (mRNA), the three mutant genes are connected to each other by 2A sequences, as shown in FIG. 2.

EXAMPLE 4

Completion of Multi-Cistronic Vector of pTet Retrovirus

(36) A psCMV vector in which a PDGF promoter and three mutant genes are connected in tandem was constructed, and was moved to the retroviral vector of Example 1 using restriction enzymes SwaI, PAcI and NotI, to complete a final recombinant expression vector. The completed recombinant expression vector was used to verify a deoxyribonucleic acid (DNA) sequence of 14,618 bp in total through a determination of a base sequence.

(37) FIG. 3A illustrates a one-dimensional (1D) structure of a multi-cistronic vector of a pTet retrovirus manufactured so that an hAPP gene, an hTau gene and a PSEN1 gene are expressed using a CMV enhancer and an hPDGF- promoter, and FIG. 3B illustrates a cyclic structure of the multi-cistronic vector. As an example of the recombinant expression vector, a base sequence of pTet-CMVE-hPDGFb-APPsw-2A-Tau-2A-PS1 may be defined with reference to SEQ ID NO: 9 described in the accompanying sequence list.

EXAMPLE 5

Confirmation of Expression of Final Recombinant Expression Vector

(38) A DNA preparation of the final recombinant expression vector was performed, a transfection of HT22 cells was performed using a Lipofectamine (Invitrogen), and whether three genes are simultaneously expressed in a cell was determined after 18 hours.

(39) Expression of the three genes was confirmed based on a western blot scheme using an antibody of each of the three genes. Expression of an APP was detected based on antibodies 22C11 and 6E10. Expression of a tau protein was detected based on an antibody Tau5, and expression of a PS1 was detected based on a PS1-specific antibody.

(40) After transient expression of the multi-cistronic vector of the pTet retrovirus in HEK-293 cells, a protein was detected using the western blot scheme. A 2A system properly operated in the recombinant expression vector.

EXAMPLE 6

Development of Pig Cell Line into Which AD Mutant Gene is Introduced

(41) A DNA preparation of the final recombinant expression vector was performed, a transfection of ear cells of a pig was performed using an electroporation, and cell lines were selected by hygromycin. The electroporation was performed under a total of 11 conditions.

(42) TABLE-US-00003 TABLE 3 3275 set parameters Poring Pulse (PP) Transfer Pulse (TP) Decay Decay Time Interval Number rate Time Interval Number rate # V (ms) (ms) of times (%) V (ms) (ms) of times (%) Polarity 1 Control group (with cells and DNA on which electroporation is not performed) 2 125 2.5 50 2 10 20 50 50 5 40 +/ 3 125 5 50 2 10 20 50 50 5 40 +/ 4 150 5 50 2 10 20 50 50 5 40 +/ 5 175 5 50 2 10 20 50 50 5 40 +/ 6 200 5 50 2 10 20 50 50 5 40 +/ 7 225 5 50 2 10 20 50 50 5 40 +/ 8 275 0.5 50 2 10 20 50 50 5 40 +/ 9 275 1 50 2 10 20 50 50 5 40 +/ 10 275 1.5 50 2 10 20 50 50 5 40 +/ 11 275 2 50 2 10 20 50 50 5 40 +/

(43) Table 3 shows conditions of the electroporation.

(44) Cell lines were selected by continuously processing hygromycin (300 g/mL) for five days under condition #5 (175 V, 5 ms) with a highest efficiency of expression and gene introduction. In the selected cell lines, whether three mutant genes were introduced into a chromosome was determined using a PCR.

(45) FIG. 4 illustrates primers used in a PCR to verify integration of a multi-cistronic vector of a pTet retrovirus.

(46) FIG. 5 illustrates 16 cell lines of a pig using a multi-cistronic vector of a pTet retrovirus.

(47) Cell lines were acquired from 16 colonies in total, respectively. It is found from FIG. 5 that all gene sites including a promoter site, mutant gene sites and a poly A site are integrated in a chromosome.

EXAMPLE 7

Analysis of Pig Cell Line into AD Mutant Gene is Introduced

(48) The cell lines of Example 6 into which AD mutant genes are introduced were subcultured to increase a number of cells. By separating the cells, a stock was formed. Proteins were extracted from produced cell lines, and whether all three AD genes are expressed was determined.

(49) Expression was detected based on the western blot scheme using the same antibodies as those used in Example 5.

(50) It is found that all three proteins are expressed in the cell lines despite a difference in an amount of proteins to be expressed.

EXAMPLE 8

Production of AD Model Pig by Nuclear Substitution or DNA Injection

(51) A fertilized egg transformed for production of an AD model pig was produced by a nuclear substitution or a DNA injection. To produce the fertilized egg using the nuclear substitution, a nucleus of an egg of a pig matured in vitro was removed from the egg, a transgenic cell containing an introduced AD gene directly was injected into the egg from which the nucleus was removed, and a cell fusion was performed in a solution of 0.3M mannitol (Sigma) using an LF201 Electro Cell Fusion Generator (NEPA GENE, Shioyaki, Japan) (at 120 volts (V) and 1 pulse 60 microseconds (s)). Whether cells are fused was observed after 30 minutes, and the cells were processed for five hours in a culture medium, that is, a porcine zygote medium (PZM)-5 to which 7.5 mg/ml of cytochalasin B (sigma) was added.

(52) To produce the fertilized egg using the DNA injection, an egg of a pig matured in vitro was fertilized in vitro by a fresh sperm of a pig for five hours (at a sperm concentration of 5,000 sperms per egg). When 18 hours have elapsed after the fertilization, fat globules were collected to one side by performing a centrifugation at a rate of 15,000 to 18,000 revolutions per minute (rpm) for ten minutes, and were fixed by a micromanipulator. A transgenic DNA with a concentration of 2 ng/ul was injected into pronuclei of the fertilized egg using an eppendorf femtojet.

(53) A fertilized egg transformed using the nuclear substitution or the DNA injection was implanted into a surrogate mother, to produce an AD model pig. Typically, approximately 150 through 200 eggs were implanted per a single surrogate mother.

(54) For the implantation, a pig in a preovulatory state was used to as a recipient pig. A primary ultrasonography was performed around 100 days after the implantation. A secondary ultrasonography was performed around 150 days after the implantation, and labor was induced performed around 180 days after the implantation, to produce an AD model pig.

(55) An AD model pig born using the nuclear substitution has a weight of 568 grams (g) and a body length of 23 centimeters (cm), and is a female that is the same as a cell line.

EXAMPLE 9

Analysis of PCR to Determine Whether AD Model Pig is Transformed

(56) A PCR was performed to determine whether an AD gene is expressed in a cloned pig produced using a somatic cell into which an AD gene is introduced. Each DNA was extracted from a tissue of the cloned pig, a tissue of a surrogate mother and a donor cell. The DNA was amplified using a primer for an AD-related gene, and an electrophoresis was performed.

(57) A result obtained by analyzing whether AD-induced transformation was performed using a PCR indicates that the cloned pig has the same expression pattern as that of the donor cell and is determined as a pig transformed to induce an AD. The donor cell was used as a positive control.

EXAMPLE 10

Analysis of Parentage Test of AD Model Pig

(58) To verify that a cloned pig born using the nuclear substitution of Example 8 is derived from a donor cell, a parentage test was analyzed. DNA was extracted from a tissue sample and a cell, and a sex chromosome X and four autosomal short tandem repeat (STR) sites, that is, S0226, S0227, S0018, and SW316 with a high polymorphism for each subject, were selected. DNA of each site was amplified using oligonucleotides with a fluorescent dye by a PCR, and a fragment analysis was performed using an automatic base sequence analyzing apparatus (ABI 3130xl Genetic Analyzer, Applied Biosystems).

(59) FIG. 6 illustrates a result of a DNA fragment analysis of S0227 among STR sites of each of a surrogate mother, a donor cell and cloned pigs.

(60) FIG. 7 is a table showing a result of a DNA fragment analysis of S0227, S0018 and SW316 among STR sites of each of a surrogate mother, a donor cell and cloned pigs.

(61) The results of FIGS. 6 and 7 indicate that the clone pigs and the donor cell have the same genes in the four STR sites even though the surrogate mother shows a completely different gene pattern. Thus, it is found that a parent-child relationship between the surrogate mother and the cloned pigs is not formed and that the cloned pigs are derived from the donor cell and produced by the nuclear substitution.