KR101703506B1 - Transfection system for production of transgenic animal - Google Patents

Abstract

The present invention relates to a transplantation system for the production of transgenic animals. The transplantation system according to the present invention can be transformed directly into the adult body of an animal by using a vector for a virus, Not only the suppression of the expression of the desired gene can be controlled by changing only the shRNA sequence of the gene which is located at the desired site, but also the termination codon between the two sequences recognized by the site-specific recombinase, Expression can be inhibited. Therefore, the use of the transfusion system according to the present invention is expected to be useful for studying the role of various genes because it can inhibit the expression of a desired gene in a cell-specific manner at a desired time in various animal species.

Description

Technical Field [0001] The present invention relates to a transfection system for transgenic animals,

The present invention relates to a transfusion system for the production of transgenic animals.

Upregulation or downregulation of a specific gene is closely related to the development of disease and the development of disease. Thus, the production of transgenic animals that have artificially regulated gene expression is an essential element in studying the precise role of specific genes in the study of disease. To this end, methods for producing transgenic animals, such as non-selection methods and transposon-mediated rearrangement, have been developed. Recently, the most active method has been to use oocytes or embryonic stem cells to knock out the oocyte or embryonic stem cell gene and to amplify the gene Lt; / RTI > However, in this method, since the expression of the gene is suppressed from the early stage of development, it affects all kinds of cells, so that the survival of the transgenic animal itself is difficult, or the survival method to replace the gene whose expression is inhibited is different The expression of the genes is increased or decreased. In addition, since the transgenic animals are required to be grown into an adult through the development process or to produce a complete transgenic animal through mating, a long time, high cost And the like.

In order to overcome such a problem, recently, cell or tissue specific genetic engineering techniques have been actively developed. Recently, a Cre-recombinant-loxP system capable of regulating the expression of an organ-specific gene has been widely used (Korean Endocrinol. 2006 (21) (5): 364-639). Cre recognizes loxP locally as a recombinase and can miss out genes in selected cells or tissues. Despite these advantages, however, it still takes a long time to cross the mice, and there are still disadvantages that it is not possible to produce a desired transgenic animal after mating, and it is not applicable to other species. In addition, according to a report from the National Institutes of Health (NIH), it has been reported that the cell-specific Cre gene is introduced into embryonic stem cells and the transgenic animals cost about 100,000 to 200,000 USD (united states dollar) There is a bar.

Thus, in order to produce transgenic animals at a low cost within a short period of time, a system capable of directly regulating gene expression directly in adult animals is needed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a transfusion system for transgenic animal capable of suppressing gene expression directly in cell- It is an object of the present invention to provide a transgenic animal produced.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions, and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

As used herein, the term "cell-type specific promoter" refers to a promoter capable of regulating the expression of a sub-gene only in a desired cell type, so that the recombinant expression vector of the present invention can be used only in a desired specific cell Means a regulatable promoter. For example, the Hoxb7 promoter refers to a gene (promoter) that expresses a sub-gene only in a collecting duct cell among various kinds of cells constituting a kidney, but it can be used to regulate the expression of a vector in a cell-specific manner But are not limited thereto.

As used herein, the term "site-specific recombinase" refers to a recombinant enzyme that recognizes only a specific sequence (recognition sequence) and performs a recombination function. For example, it refers to a recombinase that recognizes two loxP sequences such as Cre, but is not limited to a recombinant enzyme that recognizes two specific sequences and removes the middle sequence.

As used herein, the term "target gene " refers to a target gene whose expression is desired to be regulated or whose function is to be confirmed.

As used herein, the term " oligonucleotide for inhibiting expression "means a nucleotide fragment capable of inhibiting the expression of a gene of interest, and includes shRNA (short hairpin RNA), siRNA (silencing RNA ), miRNA (micro RNA), antisense-oligonucleotide, etc., but it is not limited to a nucleotide fragment capable of inhibiting the expression of a target gene.

In the present specification, the term "desired timing" means a period when a function of a target gene is desired to be confirmed, and there is no limit to the timing when the vector of the present invention can be transfected. The term "function of the target gene" means the role and function of the target gene according to the timing of the target gene, the organization (by location), and the like, and is not limited thereto as long as it can be confirmed by controlling the expression of the gene.

As used herein, "vector" means a DNA fragment, nucleic acid molecule or the like that is transferred into a cell, which can replicate the DNA and can be independently re-manufactured in the host cell, Can be used interchangeably. "Expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence essential for expressing a coding sequence operably linked in a particular host organism, and is generally a selection marker used in a vector, , A fluorescent protein expression gene, and the like.

In the present specification, "transfection" means a method of directly introducing DNA into animal cells to mutate the genetic traits of the cells, and there is no limitation as long as the vector of the present invention can be introduced into cells.

As used herein, the term "transgenic animal " refers to an animal capable of artificially regulating the expression of a target gene using the transfusion system of the present invention, and includes mouse, pig, There are no restrictions on animal species such as sheep. In addition, the present invention is not limited thereto, as long as it is a plant, an insect, or the like that can regulate gene expression using the transfusion system of the present invention.

(A) transfection of a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase; And (b) transfecting a second recombinant expression vector comprising the recognition sequence of the site-specific recombinase and an oligonucleotide for inhibiting expression of the target gene, wherein the second recombinant expression vector comprises two The present invention provides a method for inhibiting the expression of a target gene, the method comprising a stop codon sequence between the recombinase recognition sequences. The method of inhibiting the expression of the target gene is preferably a method of eliminating a stop codon existing between two recombinant enzyme recognition sequences using a recombinant enzyme, thereby suppressing the expression of the target gene through RNA interference.

(A) transfection of a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase; And (b) transfecting a second recombinant expression vector comprising a recognition sequence of the site-specific recombinase and an oligonucleotide for inhibiting expression of the target gene at a desired time, and the second recombinant The expression vector provides a method for identifying the function of a target gene at a desired timing, including a stop codon sequence between two recombinase recognition sequences.

(A) a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase; And (b) a second recombinant expression vector comprising the recognition sequence of the site-specific recombinase and an oligonucleotide for inhibiting the expression of the target gene, wherein the second recombinant expression vector comprises two recombinant enzyme recognition sequences The present invention provides a composition for inhibiting the expression of a target gene, the composition comprising a stop codon sequence between the target gene and the target gene.

The present invention also provides a recombinant vector comprising (a) a first region comprising a cell-type specific promoter and a site-specific recombinase; And (b) a second region comprising a promoter, a recognition sequence for the site-specific recombinase, and an oligonucleotide for suppressing expression of the target gene. In addition, the present invention provides a method for producing a transgenic animal, excluding human, comprising transfection of the vector into an animal other than a human, and a transgenic animal produced by the method. In one embodiment of the present invention, the recombinant expression vector is preferably a vector described in Fig.

(A) transfection of an animal, except human, with a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase; ; And (b) transfecting an animal other than a human with a second recombinant expression vector comprising a recognition sequence for the site-specific recombinase and an oligonucleotide for inhibiting the expression of the target gene, The recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences.

(A) a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase; And (b) a second recombinant expression vector comprising the recognition sequence of the site-specific recombinase and an oligonucleotide for inhibiting the expression of the target gene, wherein the second recombinant expression vector comprises two recombinant enzyme recognition sequences The present invention provides a transgenic animal, excluding human, comprising a stop codon sequence.

In one embodiment of the present invention, the cell-specific promoter is preferably a Hoxb7 promoter including the nucleotide sequence of SEQ ID NO: 15, but is not limited thereto as long as it is a cell-specific expression-regulating promoter.

In another embodiment of the present invention, the recombinant enzyme can be regulated only in a specific cell under the control of the cell-specific promoter, preferably Cre comprising the nucleotide sequence of SEQ ID NO: 16, A Flp including a nucleotide sequence or the like, or a recombinant enzyme capable of recognizing a specific sequence and removing the gene between specific sequences.

In another embodiment of the present invention, the first recombinant expression vector is preferably a vector as described in FIG. 1A.

In another embodiment of the present invention, the sequence recognized by the position-specific recombinase is loxP comprising the nucleotide sequence of SEQ ID NO: 18, FRT including the nucleotide sequence of SEQ ID NO: 19, or the like, The sequence is not limited thereto.

In another embodiment of the present invention, the oligonucleotide for suppressing the expression of the target gene is not limited to shRNA (short hairpin RNA) of the target gene or the like, but may be a substance capable of inhibiting the expression of a specific gene.

In another embodiment of the present invention, the second recombinant expression vector is preferably a vector as described in Fig. 1B.

In another embodiment of the present invention, the recombinant expression vector may be a lentivirus, an adenovirus, a retrovirus or the like. However, if the virus is infectious to an animal cell and can be used for transduction, But is not limited thereto.

In another embodiment of the present invention, the recombinant expression vector may further include a sequence selected from a recombinant expression vector such as a selection marker and a fluorescent protein expression gene.

The transfusion system for transgenic animal production according to the present invention can control the expression of a gene of interest in a desired cell of an adult animal by changing only the cell specific promoter sequence of the vector and the shRNA sequence of the desired gene By including the termination codon between two sequences recognized by the site-specific recombinase, gene expression can be inhibited through RNA interference at a desired time. Therefore, by using the transfusion system according to the present invention, it is possible to inhibit the expression of the target gene in a cell-specific manner at a desired time in various animal species, so that a transgenic animal can be produced at a low cost with high efficiency in a short period of time Therefore, it is expected that it can be used easily in studying the roles of various genes.

1 is a diagram illustrating a dual trait injection system according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of checking the efficiency of a transfusion system in vitro according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing a result of checking the efficiency of a single trait injection system in vivo according to an embodiment of the present invention.
FIG. 4 is a view showing the results of checking the efficiency of a double-transduction system in vivo according to an embodiment of the present invention.
FIG. 5 is a graph showing a result of checking the efficiency of a double transfusion system in vivo according to an embodiment of the present invention. FIG.
6 is a diagram illustrating a single vector system in accordance with an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1: Production of transfusion system

1.1. Production of vector

Lentiviruses expressing loxP-AQP3 shRNA and lentiviruses expressing HoxB7-Cre were prepared in order to construct a transfection system for producing transgenic animals . First, in order to construct a vector for lentivirus, siRNA Selection Web Server (http://jura.wi.mit.edu/) based on the mouse aquaporin-3 (AQP3) cDNA sequence The siRNA target site was determined in the bioc / siRNA (siRNA), and a scrambled sequence was used as the control. Each synthesized oligonucleotide was inserted into the XhoI-HpaI site of the pSico lentiviral vector (Addgene). The nucleotide sequences (SEQ ID NOS: 1 to 4) are shown in Table 1. In addition, the HoxB7 promoter was inserted into the XbaII-NheI site of the Puro-Cre empty vector (Addgene). Each fabricated vector design is shown in Fig.

For the single vector system, the sequence located before the U6 promoter of the "loxP-shAQP3" vector was cut with restriction enzyme (XbaI), and a single vector was constructed by inserting the Hoxb7 promoter-Cre recombinase sequence (SEQ ID NO: 20). The fabricated single vector system design is shown in FIG.

Gene Sequence (5 '-> 3') SEQ ID NO: AQP3 shRNA Sense: TGCCATCGTTGACCCTTATATTCAAGAGATATAAGGGTCAACGATGGCTTTTTTC One Antisense: TCGAGAAAAAAGCCATCGTTGACCCTTATATCTCTTGAATATAAGGGTCAACGATGGCA 2 Scrambled Sense: TGCGTATGAGTAGTGCGCAGATTTCAAGAGAATCTGCGCACTACTCATACGCTTTTTTC 3 Antisense: TCGAGAAAAAAGCGTATGAGTAGTGCGCAGATTCTCTTGAAATCTGCGCACTACTCATACGCA 4

1.2. Transformed Lentivirus  making

Human embryonic kidney (HEK) 293FT cell line (Invitrogen) was inoculated with each expression vector prepared in accordance with the method of Example 1.1 and two kinds of helper plasmids, pCMV? 8.9 and water- The vesicular stomatitis virus G protein plasmid was transfected together with the calcium phosphate method. After 72 hours, the supernatant was collected, centrifuged at 780 g for 5 minutes, filtered using a 0.45-μm filter, centrifuged at 83,000 g for 1.5 hours, and the precipitated pellet was collected. To the collected pellet, 100 μL of PBS (phosphate buffered saline) was added, and the amount of lentivirus was measured. Then, the pellet was divided into ⁴ × ^{10 8} transfection units / mL and stored at -80 ° C. until use.

Example  2: Confirmation of gene expression inhibition

2.1. in vitro  Experiment

In order to confirm the gene silencing ability of the LV-Hoxb7 Cre lentivirus and LV-loxP shAQP3 lentivirus produced by the method of Example 1, the kidney collecting duct cells and the mesangial cells ) Was used. Each cell was treated with ⁴ × 10 ⁵ TU of lentivirus, cultured for 48 hours, changed into a new culture medium, and further incubated for 72 hours. The cultured cells were collected and used for the experiment. The transfection efficiency was determined by Western blotting of expression of mCherry and EGFP contained in each virus. Western blotting was performed by dissolving collected cells using a buffer solution for SDS (2% SDS, 10 mM Tris-HCl, 10% (vol / vol) glycerol, pH 6.8) and then treating the buffer solution for Laemmli sample After heating at 100 ° C for 5 minutes, proteins were separated by electrophoresis using 12% acrylamide denaturing SDS-polyacrylamide gel, and then transferred to a Hybond-ECL membrane using a Hoeffer semidry blotting apparatus. The membranes were incubated in blocking buffer A (1 × PBS, 0.1% Tween 20, 5% nonfat milk) for 1 hour at room temperature and then incubated with polyclonal antibody AQP3 (Abcam ), EGFP (Abcam), mCherry (GeneFopoeia), or beta-actin (Sigma-Aldrich), and then reacted at 4 ° C for 16 hours. The membrane was washed once with 1 × PBS containing 0.1% Tween 20 for 15 minutes, washed twice with 5 minutes to remove unbound antibody, and washed with 1: 2,000 diluted horseradish peroxidase-linked After reacting again in blocking buffer A containing donkey anti-goat IgG (Santa Cruz Biotechnology), unbound antibody was removed by washing again. The washed membrane was developed using an ECL solution. The amount of each protein was measured using TINA image software (Raytest).

In addition, the amount of AQP3 RNA was measured using a real-time polymerase chain reaction. To extract the RNA, 100 μl of RNA STAT-60 reagent (Tel-Test) was added to the collected cells, and then the cells were lysed by repeating the freezing and thawing steps three times. Then, 700 μl of RNA STAT-60 reagent was added After mixing, the mixture was allowed to stand at room temperature for 5 minutes. After adding 160 μL of chloroform, the mixture was vigorously mixed for 30 seconds, and centrifuged at 12,000 g for 15 minutes at 4 ° C. to collect only the supernatant. 400 μL of isopropanol was added to the collected supernatant, and RNA was precipitated by centrifugation at 12,000 g at 4 ° C. for 30 minutes. After washing with 70% ethanol, DEPC-treated third distilled water Lt; / RTI > After addition of 10 uM random hexanucleotide primer, 1 mM dNTP, 8 mM MgCl ₂ , 30 mM KCl, 50 mM Tris-HCl, 0.2 mM dithithreithol, 25 U RNase inhibitor and 40 U AMV reverse transcriptase (Boehringer Mannheim cDNA synthesis kit) The reaction was carried out at 30 ° C for 10 minutes and at 42 ° C for 1 hour, and the reaction was terminated by heating at 99 ° C for 5 minutes to synthesize cDNA. Real - time polymerase chain reaction was performed using the synthesized cDNA. The primer sequences used in the real-time PCR were shown in Table 2. 10 uL of SYBR Green PCR Master Kit and 5 pM of primer set were added to 25 ng of cDNA (5 uL) and polymerase chain reaction was performed using ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The polymerase chain reaction was carried out by heating for 30 minutes at 94 ° C for 30 seconds, at 60 ° C for 30 seconds, and at 72 ° C for 1 minute, followed by reaction at 72 ° C for 7 minutes. The temperature was set to increase from 60 ° C to 95 ° C to 2 ° C per minute, and the melting curve was confirmed. The amount of cDNA was measured using the comparative C _T method. The results are shown in Fig.

Gene Sequence (5 '-> 3') SEQ ID NO: AQP3 Sense: AGCCCTGGATCAAGCTGCCC 5 Antisense: TTGGCAAAGGCCCAGATTG 6 18s RNA Sense: AGTCCCTGCCCT TTGTACACA- 7 Antisense: GATCCGAGGGCCTCACTAAAC 8

As shown in FIG. 2A, mCherry was expressed in collecting duct cells treated with LV-Hoxb7 Cre, LV-Hoxb7 Cre and LV-loxPr scr, whereas EGFP was treated with LV-loxP shAQP3 Were observed only in one collecting duct cell. In addition, as shown in FIG. 2B, it was confirmed that expression of AQP3 gene was suppressed only in cells treated with LV-Hoxb7 Cre and LV-loxP shAQP3 regardless of the amount of EGFP expression. On the other hand, as shown in FIG. 2C, it was confirmed that mcherry was not expressed in the vascular septum cells, and all of the cells except LV-Hoxb7 Cre-treated cells expressed EGFP.

2.2. in vivo  Experiments (single-trait injection)

Twelve male C57BL / 6J mice were used to confirm the gene silencing ability of LV-Hoxb7 Cre lentivirus and LV-loxP shAQP3 lentivirus produced by the method of Example 1. 24 to a male C57BL / 6J mice of 26g purchased from the Jackson laboratories, and divided into four groups injected with PBS as control of 1mL, and experimental groups of 4X10 ⁸ TU-Hoxb7 Cre LV, LV-loxP shAQP3, or LV- loxP sc was transfected on days 0, 4, and 8 by hydrodynamic tail vein injection method, and each mouse was used for the experiment six weeks after the first injection. Transfection efficiency was determined by semi-nested PCR and immunofluorescence staining of each mouse liver, spleen, kidney, trachea, and colon. Respectively. Semi-nested PCR was carried out in the same manner as in Example 2.1, and the respective primer sequences used were shown in Table 3. For immunofluorescence staining, each of the extracted tissues was cut into 4-μm-height slices, placed on a slide, immersed in acetone, fixed at 4 ° C. for 10 minutes, dried for 10 minutes, For 20 minutes, treated with EGFP monoclonal antibody (Abcam) diluted 1: 1,000 and reacted at room temperature for 3 hours. After washing with PBS, unbound antibody was removed and treated with Cy2 (green) -conjugated anti-mouse IgG antibody (Research Diagnostics) and incubated for 60 minutes. The antibody was then incubated with 1: 500 diluted monoclonal antibody rabbit anti-mCherry ) Or anti-AQP3 (Abcam) and confirmed by fluorescence microscopy. The results are shown in FIG.

Gene Sequence (5 '-> 3') SEQ ID NO: EGFP Sense: GCAGCACGACTTCTTCAAGT 9 Antisense: CCGTCCTCCTTGAAGTCGAT 10 Semi-sense: GAGCGCACCATCTTCTTCA 11 mCherry Sense: ACAAGGTGAAGCTGCGCG- 12 Antisense: TTGACCTCAGCGTCGTAGTGGC 13 Semi-sense: CAGAAGAAGACCATGGGCTG 14

As shown in Fig. 3, EGFP was strongly expressed in liver, spleen and kidney, while mCherry was expressed only in kidney.

2.3. in vivo  Experiment (double transfusion)

LV-Hoxb7 Cre, LV-loxP shAQP3, LV-Hoxb7 Cre, and LV-loxP shAQP3 were transfected into C57BL / 6J mice, respectively, in order to confirm whether transgenic animals in which the expression of AQP3 was suppressed were produced normally. For dual transfection, LV-loxP shAQP3 was injected at days 3, 7, and 11 after the last day of injection of LV-HoxB7 Cre. Then, western blotting and real-time PCR were performed in the same manner as in Example 2.1, the urine of the mice was collected for 24 hours before the end of the experiment, the volume of the urine was measured, and a vapor pressure osmometer ; Wescor) was used to measure osmolality. The results are shown in Fig. 4 and Fig.

As shown in FIG. 4, mCherry was expressed only in the collecting duct cell in LV-Hoxb7 Cre transfected cells, and expression of EGFP protein was increased in all cells including collecting duct cells in LV-loxP shAQP3 transgenic mice (Fig. 4C). However, in both experimental groups, it was confirmed that there was no change in the expression level of AQP3, urine volume, and osmotic pressure of urine (Fig. 4D). On the other hand, in the mice transfected with LV-Hoxb7 Cre and LV-loxP shAQP3, the amount of protein and mRNA expression of AQP3 was decreased (Fig. 4B), and the urine volume was increased and the osmotic pressure was decreased (Fig. 4D).

In addition, as shown in FIG. 5, it was confirmed that EGFP protein was expressed in the airways and colon of mice transfected with LV-Hoxb7 Cre and LV-loxP shAQP3, but the expression level of AQP3 protein and mRNA was not changed. From the above results, it was confirmed that the Hoxb7 promoter is activated only in the kidney, and that the LV-HoxB7 Cre and LV-loxP shAQP3 systems function normally in the kidney.

Through the above results, it was confirmed that the transfection system of the present invention is effectively operated both in vitro and in vivo. When the transformant system is used to convert the promoter and gene into the desired species, It was confirmed that a cell-specific gene expression-suppressing transgenic animal can be easily produced.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Transfection system for production of transgenic animal <130> DPB140051 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> AQP3 shRNA - Sense <400> 1 tgccatcgtt gacccttata ttcaagagat ataagggtca acgatggctt ttttc 55 <210> 2 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> AQP3 shRNA - Antisense <400> 2 tcgagaaaaa agccatcgtt gacccttata tctcttgaat ataagggtca acgatggca 59 <210> 3 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Scrambled - Sense <400> 3 tgcgtatgag tagtgcgcag atttcaagag aatctgcgca ctactcatac gcttttttc 59 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Scrambled - Antisense <400> 4 tcgagaaaaa agcgtatgag tagtgcgcag attctcttga aatctgcgca ctactcatac 60 gca 63 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AQP3 - Sense <400> 5 agccctggat caagctgccc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AQP3 - Antisense <400> 6 ttggcaaagg cccagattg 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 18s RNA - Sense <400> 7 agtccctgcc ctttgtacac a 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 18s RNA - Antisense <400> 8 gatccgaggg cctcactaaa c 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGFP primer - Sense <400> 9 gcagcacgac ttcttcaagt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGFP primer - Antisense <400> 10 ccgtcctcct tgaagtcgat 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EGFP primer - Semi-sense <400> 11 gagcgcacca tcttcttca 19 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mCherry primer - Sense <400> 12 acaaggtgaa gctgcgcg 18 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mCherry primer - Antisense <400> 13 ttgacctcag cgtcgtagtg gc 22 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mCherry primer - Semi-sense <400> 14 cagaagaaga ccatgggctg 20 <210> 15 <211> 1441 <212> DNA <213> Artificial Sequence <220> <223> Hoxb7 promoter <400> 15 tgttgtctgc gcctgaaaag ggcggaagag ttacaataaa gtttacaagc gagaacccga 60 gactggcccg ggccgccgct cctcattcgc tcctaggcgc cttgcagggc tgggggtggg 120 ggggagctgg tcagcaggct cctgggctgg cctaggctag gtcgctgaga ggagggggcg 180 ggggcggggg ctggaagcag gtggtgcgag tccctgggcc caggggcgca gggggtgagg 240 gaggcggctg aacgtgattg gaggagagag gatcgaggga ggggagccaa gagaaacccc 300 ctccccttgc attctgaggc tgaaggacca gggagactcc agcgcccagg ccgctcttgg 360 gaagagatct acccaggctg gtggctagtg tcccccgccg cttttctctt tgtttccgtg 420 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtttggg ggggggggta 480 cgggggggtg agaaagatgc agcgcaagac ttctgagttt ctatttccat tttttccttg 540 gactcaggtt ggggaaacag gagcagaggg aagcggttcc tccctacctc ccctctctgg 600 gacgtcgtca ctttctccca gtttctaggc ctcggcttgc cgcagccttc cttccttcgt 660 tgcttctgcc ttcctggcag ccacgctcca gtgagtgagg catccgcctt ccggaaccgg 720 gaaagcagcg agccggaccc aagcctcctt cctcccttcc tttttctccc agcccccatt 780 ccattctttt taaattttgt atatcttttt attgtcatca gaaatctcag cgtccaacgc 840 cttattgggt tggatctctg ccttagggac gccttggtca catctagtta ctacaactgg 900 ggcactaaga caaccgggag gccaggctcg cctcctcttg ggagaagagc agcagctcgg 960 atgaattaac ccaaattaat aaatattcgg ccagcaccca cccaccaagt tgcgaacatt 1020 caatccctgc gtctctctcg ctctgtaacc ggctggggga aatgggtggg ggatgacaac 1080 acggttccct cagaggttat ttattttctc ttccactcaa ttccttcttc cccaaatctc 1140 gcctgcaagc tgcctccagc ccgcgggggt cgacagcggc ccttaagccc ccagccccaa 1200 tccgcagagc tcggccttcc cattcattat tgatcatatt ttataaatcc aacgccacac 1260 aattttttcc acattactgg gagcctccgg gaggccgtca taccattggc cgaggggata 1320 tcacgtgggc cggggtcacg tggtcagaag aggaaaaagg gggtcctttt ggtgtaaatc 1380 tggactctaa ttctgtaata tatcaaggaa tctcgtaaaa ccgacactaa aacgtccccg 1440 a 1441 <210> 16 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> Cre <400> 16 aagaagagga aggtgtccaa tttactgacc gtacaccaaa atttgcctgc attaccggtc 60 gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg 120 ttttctgagc atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc 180 aagttgaata accggaaatg gtttcccgca gaacctgaag atgttcgcga ttatcttcta 240 tatcttcagg cgcgcggtct ggcagtaaaa actatccagc aacatttggg ccagctaaac 300 atgcttcatc gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt ttcactggtt 360 atgcggcgga tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg 420 ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg aaaatagcga tcgctgccag 480 gatatacgta atctggcatt tctggggatt gcttataaca ccctgttacg tatagccgaa 540 attgccagga tcagggttaa agatatctca cgtactgacg gtgggagaat gttaatccat 600 attggcagaa cgaaaacgct ggttagcacc gcaggtgtag agaaggcact tagcctgggg 660 gtaactaaac tggtcgagcg atggatttcc gtctctggtg tagctgatga tccgaataac 720 tacctgttt gccgggtcag aaaaaatggt gttgccgcgc catctgccac cagccagcta 780 tcaactcgcg ccctggaagg gatttttgaa gcaactcatc gattgattta cggcgctaag 840 gatgactctg gtcagagata cctggcctgg tctggacaca gtgcccgtgt cggagccgcg 900 cgagatatgg cccgcgctgg agtttcaata ccggagatca tgcaagctgg tggctggacc 960 aatgtaaata ttgtcatgaa ctatatccgt aacctggata gtgaaacagg ggcaatggtg 1020 cgcctgctgg aagatggcga ttag 1044 <210> 17 <211> 1258 <212> DNA <213> Artificial Sequence <220> <223> FLP <400> 17 atgccacaat ttgatatatt atgtaaaaca ccacctaagg tcctggttcg tcagtttgtg 60 gaaaggttga aagaccttca ggggaaaaaa tagcatcatg tgctgctgaa ctaacctatt 120 tatgttggat gattactcat aacggaacag caatcaagag agccacattc atgagctata 180 atactatcat aagcaattcg ctgagtttcg atattgtcaa caaatcactc cagtttaaat 240 acaagcgcaa aaagcaacaa ttctggaagc ctcattaaag aaattaattc ctgcttggga 300 atttacaatt attccttaca atggacaaaa acatcaatct gatatcactg atattgtaag 360 tagtttgcaa ttacagttcg aatcatcgga agaagcagat aagggaaata gccacagtaa 420 aaaaatgctt aaagcacttc taagtgaggg tgaaagcatc tgggagatca ctgagaaaat 480 actaaattcg tttgagtata cctcgagatt tacaaaaaca aaaactttat accaattcct 540 cttcctagct actttcatca attgtggaag attcagcgat attaagaacg ttgatccgaa 600 atcatttaaa ttagtccaaa ataagtatct gggagtaata atccagtgtt tagtgacaga 660 gacaaagaca agcgttagta ggcacatata cttctttagc gcaaggggta ggatcgatcc 720 acttgtatat ttggatgaat ttttgaggaa ttctgaacca gtcctaaaac gagtaaatag 780 gaccggcaat tcttcaagca acaaacagga ataccaatta ttaaaagata acttagtcag 840 atcgtacaac aaggctttga agaaaaatgc gccttatcca atctttgcta taaagaatgg 900 cccaaaatct cacattggaa gacatttgat gacctcattt ctgtcaatga agggcctaac 960 ggagttgact aatgttgtgg gaaattggag cgataagcgt gcttctgccg tggccaggac 1020 aacgtatact catcagataa cagcaatacc tgatcactac ttcgcactag tttctcggta 1080 ctatgcatat gatccaatat caaaggaaat gatagcattg aaggatgaga ctaatccaat 1140 tgaggagtgg cagcatatag aacagctaaa gggtagtgct gaaggaagca tacgataccc 1200 cgcatggaat gggataatat cacaggaggt actagactac ctttcatcct acataaat 1258 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> LoxP <400> 18 ataacttcgt atagtataaa ttatacgaag ttat 34 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> FRT <400> 19 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc 48 <210> 20 <211> 2488 <212> DNA <213> Artificial Sequence <220> <223> Hoxb7 promoter-Cre recombinase <400> 20 tgttgtctgc gcctgaaaag ggcggaagag ttacaataaa gtttacaagc gagaacccga 60 gactggcccg ggccgccgct cctcattcgc tcctaggcgc cttgcagggc tgggggtggg 120 ggggagctgg tcagcaggct cctgggctgg cctaggctag gtcgctgaga ggagggggcg 180 ggggcggggg ctggaagcag gtggtgcgag tccctgggcc caggggcgca gggggtgagg 240 gaggcggctg aacgtgattg gaggagagag gatcgaggga ggggagccaa gagaaacccc 300 ctccccttgc attctgaggc tgaaggacca gggagactcc agcgcccagg ccgctcttgg 360 gaagagatct acccaggctg gtggctagtg tcccccgccg cttttctctt tgtttccgtg 420 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtttggg ggggggggta 480 cgggggggtg agaaagatgc agcgcaagac ttctgagttt ctatttccat tttttccttg 540 gactcaggtt ggggaaacag gagcagaggg aagcggttcc tccctacctc ccctctctgg 600 gacgtcgtca ctttctccca gtttctaggc ctcggcttgc cgcagccttc cttccttcgt 660 tgcttctgcc ttcctggcag ccacgctcca gtgagtgagg catccgcctt ccggaaccgg 720 gaaagcagcg agccggaccc aagcctcctt cctcccttcc tttttctccc agcccccatt 780 ccattctttt taaattttgt atatcttttt attgtcatca gaaatctcag cgtccaacgc 840 cttattgggt tggatctctg ccttagggac gccttggtca catctagtta ctacaactgg 900 ggcactaaga caaccgggag gccaggctcg cctcctcttg ggagaagagc agcagctcgg 960 atgaattaac ccaaattaat aaatattcgg ccagcaccca cccaccaagt tgcgaacatt 1020 caatccctgc gtctctctcg ctctgtaacc ggctggggga aatgggtggg ggatgacaac 1080 acggttccct cagaggttat ttattttctc ttccactcaa ttccttcttc cccaaatctc 1140 gcctgcaagc tgcctccagc ccgcgggggt cgacagcggc ccttaagccc ccagccccaa 1200 tccgcagagc tcggccttcc cattcattat tgatcatatt ttataaatcc aacgccacac 1260 aattttttcc acattactgg gagcctccgg gaggccgtca taccattggc cgaggggata 1320 tcacgtgggc cggggtcacg tggtcagaag aggaaaaagg gggtcctttt ggtgtaaatc 1380 tggactctaa ttctgtaata tatcaaggaa tctcgtaaaa ccgacactaa aacgtccccg 1440 aatgaagaag aggaaggtgt ccaatttact gaccgtacac caaaatttgc ctgcattacc 1500 ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca 1560 ggcgttttct gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg 1620 gtgcaagttg aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct 1680 tctatatctt caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct 1740 aaacatgctt catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact 1800 ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct 1860 agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg 1920 ccaggatata cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc 1980 cgaaattgcc aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat 2040 ccatattggc agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct 2100 gggggtaact aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa 2160 taactacctg ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca 2220 gctatcaact cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc 2280 taaggatgac tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc 2340 cgcgcgagat atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg 2400 gaccaatgta aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat 2460 ggtgcgcctg ctggaagatg gcgattag 2488

Claims (20)

(a) transfection of a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase in a postnatal individual, excluding a human, ; And
(b) transfecting the subject with a second recombinant expression vector comprising a recognition sequence for a site-specific recombinase and an oligonucleotide for inhibiting expression of the target gene,
Wherein the second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences. The method according to claim 1,
Characterized in that the cell-specific promoter is a Hoxb7 promoter comprising the nucleotide sequence of SEQ ID NO: 15. The method according to claim 1,
Wherein said recombinant enzyme is under the control of a cell-specific promoter. The method according to claim 1,
Wherein the site-specific recombinase is Cre or Flp. The method according to claim 1,
Wherein the sequence recognized by the site-specific recombinase is loxP or FRT. The method according to claim 1,
Wherein the oligonucleotide for suppressing the expression of the target gene is shRNA (short hairpin RNA) of the target gene. The method according to claim 1,
Wherein said recombinant expression vector is for lentivirus, adenovirus, or retrovirus. (a) transfection of a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase in a postnatal individual, excluding a human, ; And
(b) transfecting a second recombinant expression vector comprising a recognition sequence of the site-specific recombinase and an oligonucleotide for inhibiting expression of the target gene at a desired time in the subject,
Wherein said second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences. 9. The method of claim 8,
Characterized in that the cell-specific promoter is a Hoxb7 promoter comprising the nucleotide sequence of SEQ ID NO: 15. 9. The method of claim 8,
Wherein said recombinant enzyme is under the control of a cell-specific promoter. 9. The method of claim 8,
Wherein the site-specific recombinase is Cre or Flp. 9. The method of claim 8,
Wherein the sequence recognized by the site-specific recombinase is loxP or FRT. 9. The method of claim 8,
Wherein the oligonucleotide for suppressing the expression of the target gene is shRNA (short hairpin RNA) of the target gene. 9. The method of claim 8,
Wherein said recombinant expression vector is for lentivirus, adenovirus, or retrovirus. A first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase, a recognition sequence of the site-specific recombinase and an oligonucleotide for suppressing the expression of the target gene a second recombinant expression vector comprising an oligonucleotide,
Wherein the second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences, wherein the second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences. (a) a first region comprising a cell-type specific promoter and a site-specific recombinase; And
(b) a second region comprising a promoter, a recognition sequence for the site-specific recombinase, and an oligonucleotide for suppressing expression of the target gene. The vector of claim 16 A method for producing a transgenic animal other than a human, comprising transfection of an animal other than a human. A vector comprising the vector of claim 16, Transgenic animals except humans. (a) transforming a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase into an animal other than a human, transfection; And
(b) transforming a second recombinant expression vector comprising an recognition sequence for a site-specific recombinase and an oligonucleotide for inhibiting the expression of a target gene into an animal other than a human,
Wherein said second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences. A first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase, a recognition sequence of the site-specific recombinase and an oligonucleotide for suppressing the expression of the target gene a second recombinant expression vector comprising an oligonucleotide,
Wherein said second recombinant expression vector comprises a stop codon sequence between two recombinase recognition sequences, as a single post-natal transgenic animal except human.

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