WO2016104906A9 - Système de transfection pour la production d'animal génétiquement modifié - Google Patents

Système de transfection pour la production d'animal génétiquement modifié Download PDF

Info

Publication number
WO2016104906A9
WO2016104906A9 PCT/KR2015/007926 KR2015007926W WO2016104906A9 WO 2016104906 A9 WO2016104906 A9 WO 2016104906A9 KR 2015007926 W KR2015007926 W KR 2015007926W WO 2016104906 A9 WO2016104906 A9 WO 2016104906A9
Authority
WO
WIPO (PCT)
Prior art keywords
expression vector
recombinant
specific
sequence
recombinant expression
Prior art date
Application number
PCT/KR2015/007926
Other languages
English (en)
Korean (ko)
Other versions
WO2016104906A1 (fr
Inventor
강신욱
Original Assignee
연세대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 연세대학교 산학협력단 filed Critical 연세대학교 산학협력단
Publication of WO2016104906A1 publication Critical patent/WO2016104906A1/fr
Publication of WO2016104906A9 publication Critical patent/WO2016104906A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/007—Vectors comprising a special translation-regulating system cell or tissue specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
    • C12N2840/102—Vectors comprising a special translation-regulating system regulates levels of translation inhibiting translation

Definitions

  • the present invention relates to a transfection system for the production of transgenic animals.
  • the upregul at i on or downregul at i on of a particular gene is closely related to the induction of disease and the development of the disease. Therefore, the production of artificially regulated transgenic animals (t ransgeni c animals) is essential for studying the exact role of specific genes in the study of disease. Transgenic animal production methods such as mediated rearrangement have been developed. Recently, the most actively used method is oocytes or embryonic stem cells (embryoni c stem eel Is), which knocks out the genes of oocytes or embryonic stem cells, It is a method of suppressing the expression of genes from the beginning of development.
  • this method affects all kinds of cells by inhibiting the expression of genes from the beginning of development, so that the survival of the transgenic animal itself is difficult, or other unexpected methods to replace the genes whose expression is suppressed. Not only does it have various problems such as increased or decreased expression of genes, but it also has a long time in the production of transgenic animals because it is necessary to produce a fully transgenic animal through maturation, or through mating. In fact, high costs are required.
  • Cre-recombinant-1 oxP system which can regulate the expression of genes by organspeci fic, has been widely used (Korean Journal of Endocrinology (2006) 21 (5): 364-639). Cre is a recombinant enzyme 0 ⁇ «) 111 ⁇ 56) that can recognize ⁇ ⁇ specifically to knock out genes in selected cells or tissues.
  • NASH National Institutes of Health
  • the present invention has been made to solve the above-mentioned problems in the prior art, a transfection system for producing a transgenic animal that can directly inhibit the expression of genes directly in adult species of various species and use thereof
  • the purpose is to provide a transgenic animal produced.
  • a recombinant expression vector of the present invention refers to a promoter that can be regulated to operate only in specific cells of interest.
  • Various types of cells that make up the kidney, such as the Hoxb7 promoter Means a gene (promoter) that expresses the subgene only in the collecting duct cell, but is not limited to any sequence that can control the expression of the vector specifically.
  • site-specific recombinase refers to a recombinant enzyme that performs recombination by recognizing only a specific sequence (recognition sequence).
  • recognition sequence a recombinant enzyme that recognizes two ⁇ sequences, such as Cre, but is not limited to any recombinant enzyme capable of recognizing two specific sequences on both sides and removing the middle sequence.
  • target gene refers to a target gene for which expression is to be regulated or to confirm function, and there is no limitation on the type of gene.
  • expression inhibiting oligonucleotide refers to a nucleotide fragment capable of suppressing the expression of a gene of interest, and specifically, a shRNA (short hairpin RNA), siRNA (siRNA) that specifically acts on a gene of interest. lencing NA), miRNA (micro RNA), antisense-oligonucleotide, etc., but may be a nucleotide fragment that can inhibit the expression of the target gene is not limited thereto.
  • the "target time” means a time to comprehensively check the function of the target gene, and there is no limitation as long as it is a step capable of transfecting the vector of the present invention.
  • the “function of the target gene” refers to the role, function, etc. of the target gene according to the time, organization (location) and the like of the target gene, if the element can be confirmed by controlling the expression of the gene is not limited thereto.
  • vector refers to a DNA fragment, nucleic acid molecule, etc. that is delivered into a cell, wherein the vector replicates DNA and can be independently remanufactured in a host cell and compatible with the term "carrier".
  • “Expression vector” means a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a particular host organism, and generally a selection marker used in the vector. ), And various base sequences such as fluorescent protein expression genes may be further included.
  • transfection means a method of directly introducing DNA into an animal cell to mutate the genetic trait of the cell, and there is no limitation as long as the vector of the present invention can be introduced into the cell.
  • transgenic animal refers to an animal that can artificially control the expression of a specific gene by using the transfection system of the present invention.
  • Mouse, pig, goat There are no restrictions on species of animals such as sheep.
  • nasal layer is not limited thereto.
  • the present invention comprises the steps of: (a) transfecting a first recombinant expression vector comprising a cell-specific promoter and a site-specific recombinase; And (b) transfecting a second recombinant expression vector comprising a recognition sequence of the position-specific recombinase and an oligonucleotide for inhibiting expression of a gene of interest.
  • a method of inhibiting expression of a gene of interest comprising a stop codon sequence between two recombinant enzyme recognition sequences.
  • the present invention comprises the steps of: (a) transfecting 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 said position-specific recombinase and an oligonucleotide for inhibiting expression of a target gene at a desired time, wherein said second recombination is performed.
  • Expression vectors provide a method for identifying the function of a target gene at a desired time, including a stop codon sequence between two recombinant enzyme recognition sequences.
  • the present invention (a) a first recombinant expression vector comprising a cell-specific promoter (cen- type specific promoter) and site-specific recombinase (site- specif ic recombinase); And (b) a second recombinant expression vector comprising the recognition sequence of said position-specific recombinase and an oligonucleotide for inhibiting expression of the gene of interest.
  • a composition for inhibiting expression of a target gene comprising a stop codon sequence between two recombinant enzyme recognition sequences.
  • the present invention provides a composition comprising: (a) a first region comprising a ceU-type specific promoter and a site-specific recombinase; And (b) a second region comprising a promoter, a recognition region of the site-specific recombinase and an oligonucleotide for inhibiting expression of a target gene.
  • the present invention also provides a method for producing a transgenic animal except for humans, which comprises transfecting the vector into an animal other than a human, and a transgenic animal prepared by the method.
  • the recombinant expression vector is preferably characterized in that the vector described in FIG.
  • the present invention (a) transfection of animals except humans with a first recombinant expression vector comprising a cell-type specific promoter and a site-specific recombinase Doing; And (b) transfecting a second recombinant expression vector comprising a recognition sequence of the position-specific recombinase and an oligonucleotide for inhibiting expression of a target gene into an animal other than a human.
  • 2 Recombinant expression vectors provide a method for the production of transgenic animals, except humans, comprising a stop codon sequence between two recombinant enzyme recognition sequences.
  • the present invention is (a) a first recombinant expression vector comprising a cell-specific promoter (ceU—type specific promoter) and site-specific recombinase; And (b) a second recombinant expression vector comprising a recognition sequence of said position-specific recombinase and an oligonucleotide for inhibiting expression of a gene of interest.
  • a transgenic animal other than human comprising a stop codon sequence between two recombinant enzyme recognition sequences.
  • the cell-specific promoter is preferably a Hoxb7 promoter, including the nucleotide sequence of SEQ ID NO: 15, and the like, as long as it is a promoter capable of regulating cell-specific expression is not limited thereto.
  • the recombinant enzyme may be regulated only in specific cells under the control of the cell-specific promoter, preferably -e comprising the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: Flp and the like, which include the nucleotide sequence of 17, and a recombinant enzyme capable of recognizing a specific sequence and removing a gene between specific sequences are not limited thereto.
  • the first recombinant expression vector is preferably characterized in that the vector described in Figure 1A.
  • the sequence-specific recombinant enzyme recognizes the sequence ⁇ comprising the nucleotide sequence of SEQ ID NO: 18, FRT including the nucleotide sequence of SEQ ID NO: 19, etc., or the recombinant enzyme is recognized If it is a specific sequence is not limited thereto.
  • the oligonucleotide for inhibiting the expression of the target gene is not limited thereto as long as it is a substance capable of inhibiting the expression of a specific gene, such as shRNA (short hai rpin RNA) of the target gene.
  • the second recombinant expression vector is preferably characterized in that the vector described in FIG.
  • the recombinant expression vector may be a lentiviral, adenovirus (adenovi rus), retrovirus (ret rovi rus) and the like, but transfected with animal cells
  • adenovirus adenovi rus
  • retrovirus ret rovi rus
  • the type of virus that can be used for the virus is not limited thereto.
  • the recombinant expression vector may further include, in addition to the sequence included in the recombinant expression vector, such as a selection marker (sel ect i on marker), a fluorescent protein expression gene.
  • the transfection system for producing a transgenic animal according to the present invention can control the inhibition of expression of a gene of interest in a target cell of an adult animal by changing only the vector-specific promoter sequence of the vector and the shRNA sequence of the gene of interest.
  • a stop codon between two sequences recognized by the site-specific recombination enzyme it is possible to suppress the expression of the gene through RNA interference at the desired time. Therefore, by using the transfection system according to the present invention, it is possible to produce a transgenic animal with high efficiency in a short period of time at a low cost since it is possible to inhibit the expression of a target gene in a cell specific time at the desired time in the adult of various species It is expected that it can be easily used to study the role of various genes.
  • FIG. 1 is a diagram showing a dual transfection system according to an embodiment of the present invention.
  • FIG. 2 is a view showing the results of confirming the efficiency of the transfection system in in vi t ro according to an embodiment of the present invention.
  • FIG 3 is a view showing the results of confirming the efficiency of a single transfection system in vivo according to an embodiment of the present invention.
  • FIG. 4 is a view showing the results of confirming the efficiency of the dual transfection system in vivo in accordance with an embodiment of the present invention.
  • Figure 5 confirms the efficiency of the dual transfection system in vivo according to an embodiment of the present invention The figure which showed the result.
  • FIG. 6 is a diagram illustrating a single vector system according to an embodiment of the present invention.
  • a lent ivirus expressing loxP-AQP3 shRNA and a lent ivirus expressing HoxB7-Cre were used.
  • Produced. First, in order to produce a lentiviral vector, based on the aquaporin-3 (3 ⁇ 1330 11-3, AQP3) cDNA sequence of the mouse, siRNA Selection Web Server (http: //jura.wi. m.edu/bioc/siRNA), si NA target sites were selected, and a scrambled sequence was used as a control, and each synthesized oligonucleotide was determined by Xho I ⁇ of pSico lentiviral vector (Addgene).
  • the base sequences (SEQ ID NOS: 1 to 4) are shown in Table 1.
  • the HoxB7 promoter was inserted into the Xball-Nhel site of the Puro-Cr ⁇ empty vector (Addgene).
  • Each fabricated vector design is shown in FIG. 1.
  • a single vector was prepared by cutting the sequence located in front of the U6 promoter of the ' ⁇ — sMQP3' vector with restriction enzyme (Xbal) and inserting the Hoxb7 promoter -Cre recombinase sequence (SEQ ID NO: 20).
  • the fabricated single vector system design is shown in FIG. 6.
  • the vesicular stomatitis virus G protein plasmid was transfected together according to the calcium phosphate method. After 72 hours, the supernatant was collected and centrifuged at 780g for 5 minutes, filtered using a 0.45um filter, and the pellet was collected by centrifugation at 83,000g for 1.5 hours.
  • LV ⁇ Hoxb7 Cre lentiviral and LV—loxP shAQP3 lentiviral produced by the method of Example 1 the collecting cells (kidney collecting duct cell) and mesential cells (mesangial cell) ) was used. Each cell was treated with lentiviral 4 ⁇ 10 5 TU, incubated for 48 hours, then changed to a new culture medium, and further cultured for 72 hours, and then cultured cells were collected and used for the experiment. Transfection efficiency was measured by Western blotting of the expression level of mCherry and EGFP contained in each virus.
  • Western blotting was performed by lysing the collected cells using SDS sample buffer (2% SDS, 10 mM Tris-HCl, 10% (vol / vol) glycerol, pH6.8), and then buffer solution for Lae ⁇ ⁇ sample. After heating at 100 ° C for 5 minutes, the protein was separated by electrophoresis using a 12% aery 1 amide denaturing SDS-polyacrylamide gel, and then using a Hoeffer semi dry blotting apparatus, a Hybond-ECL membrane (membrane). ).
  • the protein-transferred membrane was polyclonal, diluted in blocking buffer A (1X PBS, 0.1% Tween 20, 5% nonfat milk), reacted for 1 hour at room temperature, and then diluted 1: 1, 000.
  • Membranes finished with reaction were washed once for 15 minutes using IX PBS added with 0.1% Tween 20, washed twice for 5 minutes to remove unbound antibody, and horseradish peroxidase-1 diluted 1: 2,000.
  • RNA STAT-60 reagent Te ⁇ Test
  • 700uL RNA STAT ⁇ 60 reagent After additional addition, mix well and leave for 5 minutes at-.
  • 160uL of chloroform (chloroform) was added and mixed vigorously for 30 seconds, and then centrifuged at 12,000g, 4 ° C for 15 minutes to collect only the supernatant.
  • Polymerase chain reaction is then at 95 ° C initially heated 9 minutes, 30 seconds at 94.5 ° C, 30 sec at 60 ° C, after repeating 35 cycles of 1 minute sequence at 72 ° C, 7 min at 72 ° C The reaction was carried out. And the temperature was set to increase to 2 ° C per minute from 60 ° C to 95 ° C, the melting curve was confirmed, and the amount of cDNA was measured using the comparative CT method. The results are shown in FIG.
  • Ant i sense GATCCGAGGGCCTCACTAAAC 8
  • mCherry was expressed in the collection tube cells treated with LV ⁇ Hoxb7 Cre, and the collection tube cells treated with LV-Hoxb7 Cre and LV-loxP scr, whereas EGFP It was confirmed that only observed in the collection tube cells treated with LV-loxP shAQP3.
  • FIG. 2B it was confirmed that expression of the AQP3 gene was suppressed only in cells treated with LVHoxb7 Cre and LV—loxP shAQP3 regardless of the amount of EGFP expression.
  • FIG. 2C mcherry was not expressed in vascular mesenteric cells, and all of the cells treated with LV-Hoxb7 Cre were expressed in EGFP.
  • Transfection efficiency was confirmed by semi-nested PCR and immunofluorescence staining by extracting the liver, spleen, kidney, trachea, and colon of each mouse. It was.
  • Semi- nested PCR was carried out in the same manner as in Example 2.1, each primer sequence used is shown in Table 3.
  • each extracted tissue was cut into 4 ⁇ height units, placed on a slide ( sl ide ), and acetone.
  • EGFP was strongly expressed in the liver, spleen, and kidney, whereas n herry was expressed only in the kidney.
  • LV57X7 Cre, LV-loxP shAQP3, LV-Hoxb7 Cre and LV-loxP shAQP3 were transfected into C57BL / 6J mice, respectively.
  • LV-loxP shAQP3 was injected on days 3, 7, and 11 after the last day of LV ⁇ HoxB7 Cre injection.
  • mice transfected with LV-Hoxb7 Cre As shown in FIG. 4, mCherry was expressed only in the collection tube cells in mice transfected with LV-Hoxb7 Cre, and expression of EGFP protein was increased in all cells including the collection tube cells in mice transfected with LV-loxP shAQP3. Confirmation (FIG. 4C). However, in both groups, there was no change in the expression level, urine volume, and osmotic pressure of urine (FIG. 4D). On the other hand, mice transfected with LV—Hoxb7 Cre and LV-loxP shAQP3 showed decreased protein and mRNA expression levels of AQP3 (FIG. 4B), increased urine volume, and decreased osmotic pressure (FIG. 4D). .
  • transfection system By using the transfection system according to the present invention, it is possible to produce a transgenic animal with high efficiency in a short period of time at a low cost since it is possible to inhibit the expression of a target gene in a specific time at a desired time in various species of animal adult. As such, it is expected to be easily used to study the role of various genes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Veterinary Medicine (AREA)

Abstract

La présente invention concerne un système de transfection pour la production d'un animal génétiquement modifié. A l'aide du système de transfection selon la présente invention, un vecteur viral peut être utilisé afin d'effectuer une modification génétique directe chez un animal adulte, de sorte que, non seulement il est possible de réguler de manière spécifique des cellules l'inhibition de l'expression d'un gène cible en modifiant uniquement la séquence promoteur spécifique de cellule du vecteur et la séquence shARN du gène cible, mais il est également possible d'inhiber l'expression génique à un moment désiré en incluant un codon de terminaison entre deux séquences reconnues par une enzyme de recombinaison spécifique de site. Par conséquent, il est prévu que le système de transfection, selon la présente invention, peut être facilement utilisé dans des études dans les rôles de différents gènes, du fait que le système de transfection est propre à inhiber de manière spécifique des cellules l'expression d'un gène cible à un moment souhaité dans diverses espèces d'animaux adultes, en utilisation.
PCT/KR2015/007926 2014-12-23 2015-07-29 Système de transfection pour la production d'animal génétiquement modifié WO2016104906A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020140186879A KR101703506B1 (ko) 2014-12-23 2014-12-23 형질전환동물 제작을 위한 형질주입 시스템
KR10-2014-0186879 2014-12-23

Publications (2)

Publication Number Publication Date
WO2016104906A1 WO2016104906A1 (fr) 2016-06-30
WO2016104906A9 true WO2016104906A9 (fr) 2016-10-20

Family

ID=56150895

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/007926 WO2016104906A1 (fr) 2014-12-23 2015-07-29 Système de transfection pour la production d'animal génétiquement modifié

Country Status (2)

Country Link
KR (1) KR101703506B1 (fr)
WO (1) WO2016104906A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102070763B1 (ko) * 2018-08-21 2020-01-29 성균관대학교산학협력단 도파민 신경세포 특이적 발현 제어 시스템
KR102479490B1 (ko) * 2019-11-13 2022-12-21 주식회사 마크로젠 신경세포 특이적 프로모터 및 이를 포함하는 dna 변형 유도용 재조합 벡터

Also Published As

Publication number Publication date
WO2016104906A1 (fr) 2016-06-30
KR101703506B1 (ko) 2017-02-07
KR20160076628A (ko) 2016-07-01

Similar Documents

Publication Publication Date Title
US11753641B2 (en) Structurally designed shRNAs
WO2017062754A1 (fr) Compositions et procédés pour augmenter l'activité crispr par inhibition de polq
US20070155014A1 (en) Methods for increasing efficiency of homologous recombination
TWI516592B (zh) 用於在原核細胞內使用真核第二型聚合酶啟動子驅動之轉錄作用的可誘導之基因表現組成物及其應用
WO2021226151A2 (fr) Sélection par knock-in d'un gène essentiel
WO2019086007A1 (fr) Arnguidecourt pour cibler et guider une protéine cas9 pour cliver efficacement des loci de gène tcr et b2m
WO2013109713A1 (fr) Ciblage d'arn à des microvésicules
JP4517061B2 (ja) ダンベル型ｄｎａの効率的な製造方法
WO2016104906A9 (fr) Système de transfection pour la production d'animal génétiquement modifié
WO2020093575A1 (fr) Polynucléotide destiné au traitement de tumeurs et son utilisation
WO2020128006A1 (fr) Miarn destiné à être utilisé en thérapie
CN110511933B (zh) 一种大鼠长链非编码lncRNA-lncMSTRG10078及其抵抗细胞损伤的应用
JP2006500017A (ja) ＲＮＡ発現のためのアデノウイルスのＶＡ１ＰｏｌＩＩＩ発現システム
KR101048417B1 (ko) 마우스의 프리온 유전자에 대한 마이크로 ｒｎａ 및 프리온유전자의 발현이 녹다운된 마우스 세포
CN118652933A (zh) Cd36基因过表达的山羊乳腺上皮细胞的构建方法及应用
US20230293690A1 (en) 5' s/mar applications
KR20240011184A (ko) Ciita 표적화 아연 핑거 뉴클레아제
CN118703565A (zh) Ltf基因过表达的山羊乳腺上皮细胞的构建方法及应用
Korostowski Transcript Regulation within the Kcnq1 Domain
Ahiable Cloning Candidate Novel Cell-Fate Genes (Pre-Implantation Mouse Development)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15873434

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15873434

Country of ref document: EP

Kind code of ref document: A1