KR20160076628A - Transfection system for production of transgenic animal - Google Patents
Transfection system for production of transgenic animal Download PDFInfo
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Abstract
Description
본 발명은 형질전환동물 제작을 위한 형질주입 시스템에 관한 것이다.The present invention relates to a transfusion system for the production of transgenic animals.
특정 유전자의 과다발현(upregulation) 또는 발현억제(downregulation) 현상은 대부분 질병의 유발 및 질병의 발달 과정과 밀접하게 연관되어져 있다. 따라서, 유전자의 발현을 인위적으로 조절한 형질전환동물(transgenic animal)들의 제작은 질병의 연구에 있어서 특정 유전자의 정확한 역할 연구를 위해 필수적인 요소이다. 이를 위하여, 비-선별 방법, 트랜스포존-매개 재배치 등 형질전환동물 제작 방법이 개발되고 있다. 최근에 가장 활발히 이용되고 있는 방법은 난모세포(oocytes) 또는 배아줄기세포(embryonic stem cells)를 이용한 방법으로, 난모세포 또는 배아줄기세포의 유전자를 낙아웃(knock-out)시켜, 발생 초기부터 유전자의 발현을 억제시키는 방법이다. 그러나 이러한 방법의 경우에는 발생 초기부터 유전자의 발현을 억제시킴으로써 모든 종류의 세포에 영향을 주기 때문에 형질전환동물의 생존 자체가 어렵거나, 발현이 억제된 유전자를 대체하기 위한 생존방식으로 예상하지 못했던 다른 유전자들의 발현이 증가되거나 감소되는 등의 다양한 문제점을 가지고 있을 뿐만 아니라, 발생과정을 거쳐 성체로 성장시키거나, 또는 교배를 통하여 완전한 형질전환동물을 제작해야 하기 때문에 형질전환동물의 제작에 장시간, 고비용이 요구되고 있는 실정이다.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.
이와 같은 문제점을 극복하기 위하여, 최근에는 세포 또는 조직 특이적인 유전자 조작 기술이 활발히 개발되고 있다. 최근에는 기관특이적(organ-specific)으로 유전자의 발현을 조절할 수 있는 Cre-recombinant-loxP 시스템이 널리 사용되어지고 있다(대한내분비학회지 (2006) 21(5): 364-639). Cre는 재조합 효소(recombinase)로서 위치 특이적으로 loxP를 인식하여 선택된 세포 또는 조직에서 유전자를 낙아웃시킬 수 있다. 그러나 이러한 장점에도 불구하고, 여전히 마우스를 교배시키는 데는 장시간이 소요될 뿐만 아니라, 교배 후 원하는 형질전환동물이 생산되는 데는 낮은 성공률을 나타내고 있고, 다른 종에는 적용할 수 없다는 단점들이 여전히 존재하고 있다. 또한, 국립보건원(NIH)의 연구 보고에 따르면, 세포특이적인 Cre 유전자를 배아줄기세포에 도입하고, 형질전환동물을 제조하는 데는 대략 10만 내지 20만 USD(united states dollar)가 소요된다고 보고된 바 있다.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.
이와 같이, 저비용으로 단기간 내에 형질전환동물을 제작하기 위해서는 성체 동물에서 직접 세포특이적으로 유전자의 발현을 조절할 수 있는 시스템이 필요한 실정이다.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.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로, 다양한 종의 성체 동물에서 직접 세포특이적으로 유전자의 발현을 억제시킬 수 있는 형질전환동물 제작을 위한 형질주입 시스템 및 이를 이용하여 제작된 형질전환동물을 제공하는 것을 그 목적으로 한다.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.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.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.
이하, 본원에 기재된 다양한 구체예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세사항, 예컨대, 특이적 형태, 조성물, 및 공정 등이 기재되어 있다. 그러나, 특정의 구체예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구체예" 또는 "구체예"에 대한 본 명세서 전체를 통한 참조는 구체예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구체예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구체예에서" 또는 "구체예"의 상황은 반드시 본 발명의 동일한 구체예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구체예에서 어떠한 적합한 방법으로 조합될 수 있다.
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.
본 명세서에 있어서, "세포특이적 프로모터(cell-type specific promoter)"란 목적하는 세포의 종류에서만 하위 유전자의 발현을 조절할 수 있는 프로모터로서, 본 발명의 재조합 발현 벡터가 목적하는 특정 세포에서만 작동되도록 조절할 수 있는 프로모터를 의미한다. 예를 들어, Hoxb7 프로모터와 같이 신장(kidney)을 구성하는 다양한 종류의 세포 중 집합관 세포(collecting duct cell)에서만 하위 유전자를 발현시키는 유전자(프로모터)를 의미하나, 세포특이적으로 벡터의 발현을 조절할 수 있는 서열이라면 이에 제한되지 않는다.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.
본 명세서에 있어서, "위치특이적 재조합 효소(site-specific recombinase)"란 특정 서열만을 인식(인식 서열)하여 재조합 작용을 하는 재조합 효소를 의미한다. 예를 들어, Cre와 같이 두 개의 loxP 서열을 인식하는 재조합 효소를 의미하나, 양 쪽 두 개의 특정 서열을 인식하여 가운데 서열을 제거할 수 있는 재조합 효소라면 이에 제한되지 않는다.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.
본 명세서에 있어서, "목적 유전자(target gene)"란 발현을 조절하고자 하는 또는 기능을 확인하고자 하는 타겟 유전자를 의미하며, 유전자의 종류에는 제한이 없다.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.
본 명세서에 있어서, "발현 억제용 올리고뉴클레오티드(oligonucleotide)"란 목적 유전자의 발현을 억제할 수 있는 뉴클레오티드 단편을 의미하며, 목적 유전자에 특이적으로 작용하는 shRNA(short hairpin RNA), siRNA(silencing RNA), miRNA(micro RNA), antisense-oligonucleotide 등 일 수 있으나, 목적 유전자의 발현을 억제할 수 있는 뉴클레오티드 단편이라면 이에 제한되지 않는다.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.
본 명세서에 있어서, "목적하는 시기"란 목적 유전자의 기능을 확인하고자 하는 시기를 포괄적으로 의미하며, 본 발명의 벡터를 형질주입할 수 있는 단계라면 시기에는 제한이 없다. 상기 "목적 유전자의 기능"이란 목적 유전자의 시기별, 조직별(위치별) 등에 따른 목적 유전자의 역할, 기능 등을 의미하며, 유전자의 발현을 조절하여 확인할 수 있는 요소라면 이에 제한되지 않는다.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.
본 명세서에 있어서, "벡터(vector)"란 세포 내로 전달되는 DNA 단편, 핵산 분자 등을 의미하며, 상기 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재제조될 수 있으며, 용어 "전달체"와 호환하여 사용될 수 있다. "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미하며, 일반적으로 벡터에 사용되는 선별 마커(selection marker), 형광 단백질 발현 유전자 등 다양한 염기서열을 추가로 포함할 수 있다.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.
본 명세서에 있어서, "형질주입(transfection)"이란, 동물 세포에 DNA를 직접 도입하여 세포의 유전 형질을 변이시키는 방법을 의미하며, 본 발명의 벡터를 세포에 도입할 수 방법이라면 제한이 없다.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.
본 명세서에 있어서, "형질전환동물(transgenic animal)"이란, 본 발명의 형질주입 시스템을 이용하여 특정 유전자(target gene)의 발현을 인위적으로 조절할 수 있는 동물을 의미하며, 마우스, 돼지, 염소, 양 등 동물의 종에는 제한이 없다. 이 외에도 식물체, 곤충 등 본 발명의 형질주입 시스템을 이용하여 유전자의 발현을 조절할 수 있는 개체라면 이에 제한되지 않는다.
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.
본 발명은 (a) 세포특이적 프로모터(cell-type specific promoter) 및 위치특이적 재조합 효소(site-specific recombinase)를 포함하는 제 1 재조합 발현 벡터를 형질주입(transfection)하는 단계; 및 (b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 형질주입하는 단계를 포함하고, 상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 목적 유전자의 발현을 억제하는 방법을 제공한다. 상기 목적 유전자의 발현을 억제하는 방법은 바람직하게는 두 개의 재조합 효소 인식 서열 사이에 존재하는 종결코돈을 재조합 효소를 이용하여 제거하고, 이를 통하여 목적 유전자의 발현을 RNA 간섭을 통해 억제하는 방법이다.
(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.
또한, 본 발명은 (a) 세포특이적 프로모터(cell-type specific promoter) 및 위치특이적 재조합 효소(site-specific recombinase)를 포함하는 제 1 재조합 발현 벡터를 형질주입(transfection)하는 단계; 및 (b) 목적하는 시기에 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 형질주입하는 단계를 포함하고, 상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 목적하는 시기의 목적 유전자의 기능을 확인하는 방법을 제공한다.
(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.
또한, 본 발명은 (a) 세포특이적 프로모터(cell-type specific promoter) 및 위치특이적 재조합 효소(site-specific recombinase)를 포함하는 제 1 재조합 발현 벡터; 및 (b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 포함하고, 상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 목적 유전자의 발현 억제용 조성물을 제공한다.
(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.
또한, 본 발명은 (a) 세포특이적 프로모터(cell-type specific promoter) 및 위치특이적 재조합 효소(site-specific recombinase)를 포함하는 제 1 지역(region); 및 (b) 프로모터, 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 지역을 포함하는 재조합 발현 벡터를 제공한다. 또한, 본 발명은 상기 벡터를 인간을 제외한 동물에 형질주입(transfection)하는 단계를 포함하는 인간을 제외한 형질전환동물의 제조 방법 및 상기 방법으로 제조된 형질전환동물을 제공한다. 본 발명의 일 구체예에서, 상기 재조합 발현 벡터는 바람직하게는 도 6에 기재된 벡터인 것을 특징으로 한다.
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.
또한, 본 발명은 (a) 세포특이적 프로모터(cell-type specific promoter) 및 위치특이적 재조합 효소(site-specific recombinase)를 포함하는 제 1 재조합 발현 벡터를 인간을 제외한 동물에 형질주입(transfection)하는 단계; 및 (b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 인간을 제외한 동물에 형질주입하는 단계를 포함하고, 상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 인간을 제외한 형질전환동물의 제조 방법을 제공한다.
(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.
또한, 본 발명은 (a) 세포특이적 프로모터(cell-type specific promoter) 및 위치특이적 재조합 효소(site-specific recombinase)를 포함하는 제 1 재조합 발현 벡터; 및 (b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 포함하고, 상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 인간을 제외한 형질전환동물을 제공한다.
(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.
본 발명의 일 구체예에서, 상기 세포특이적 프로모터는 바람직하게는 서열번호 15의 염기서열을 포함하는 Hoxb7 프로모터 등이나, 세포특이적으로 발현을 조절할 수 있는 프로모터라면 이에 제한되지 않는다.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.
본 발명의 다른 구체예에서, 상기 재조합 효소는 상기 세포특이적 프로모터의 조절 하에 있어서, 특정 세포에서만 발현이 조절될 수 있으며, 바람직하게는 서열번호 16의 염기서열을 포함하는 Cre, 서열번호 17의 염기서열을 포함하는 Flp 등이나, 특정 서열을 인식하여 특정 서열 사이 유전자를 제거할 수 있는 재조합 효소라면 이에 제한되지 않는다.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.
본 발명의 또 다른 구체예에서, 상기 제 1 재조합 발현 벡터는 바람직하게는 도 1A에 기재된 벡터인 것을 특징으로 한다.In another embodiment of the present invention, the first recombinant expression vector is preferably a vector as described in FIG. 1A.
본 발명의 또 다른 구체예에서, 상기 위치특이적 재조합 효소가 인식하는 서열은 서열번호 18의 염기서열을 포함하는 loxP, 서열번호 19의 염기서열을 포함하는 FRT 등이나, 재조합 효소가 인식하는 특정 서열이라면 이에 제한되지 않는다.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.
본 발명의 또 다른 구체예에서, 상기 목적 유전자의 발현 억제용 올리고뉴클레오티드는 목적 유전자의 shRNA(short hairpin RNA) 등이나, 특정 유전자의 발현을 억제할 수 있는 물질이라면 이에 제한되지 않는다.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.
본 발명의 또 다른 구체예에서, 상기 제 2 재조합 발현 벡터는 바람직하게는 도 1B에 기재된 벡터인 것을 특징으로 한다.In another embodiment of the present invention, the second recombinant expression vector is preferably a vector as described in Fig. 1B.
본 발명의 또 다른 구체예에서, 상기 재조합 발현 벡터는 렌티바이러스(lentivirus), 아데노바이러스(adenovirus), 레트로바이러스(retrovirus) 등 일 수 있으나, 동물 세포에 감염되어 형질주입에 사용될 수 있는 바이러스 종류라면 이에 제한되지 않는다.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.
본 발명의 또 다른 구체예에서, 상기 재조합 발현 벡터는 이외에 선별 마커(selection marker), 형광단백질 발현 유전자 등 일반적으로 재조합 발현 벡터에 포함되는 서열 등을 추가로 포함할 수 있다.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.
본 발명에 따른 형질전환동물 제작을 위한 형질주입 시스템은 벡터의 세포특이적 프로모터 서열 및 목적하는 유전자의 shRNA 서열만 변경함으로써, 성체 동물의 목적하는 세포에서 목적하는 유전자의 발현 억제를 조절할 수 있을 뿐만 아니라, 위치특이적 재조합 효소가 인식하는 두 개의 서열 사이에 종결코돈을 포함함으로써, 원하는 시기에 RNA 간섭을 통하여 유전자의 발현을 억제시킬 수 있다. 따라서 본 발명에 따른 형질주입 시스템을 이용하면, 다양한 종의 동물 성체에서 원하는 시기에 세포특이적으로 목적 유전자의 발현을 억제시킬 수 있기 때문에 저렴한 비용으로 단기간에 높은 효율로 형질전환동물을 제작할 수 있기 때문에 다양한 유전자의 역할 연구에 용이하게 사용될 수 있을 것으로 기대된다.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.
도 1은 본 발명의 일 실시예에 따른 이중 형질주입 시스템을 나타낸 도면이다.
도 2는 본 발명의 일 실시예에 따른 in vitro에서 형질주입 시스템의 효율을 확인한 결과를 나타낸 도면이다.
도 3은 본 발명의 일 실시예에 따른 in vivo에서 단일 형질주입 시스템의 효율을 확인한 결과를 나타낸 도면이다.
도 4는 본 발명의 일 실시예에 따른 in vivo에서 이중 형질주입 시스템의 효율을 확인한 결과를 나타낸 도면이다.
도 5는 본 발명의 일 실시예에 따른 in vivo에서 이중 형질주입 시스템의 효율을 확인한 결과를 나타낸 도면이다.
도 6은 본 발명의 일 실시예에 따른 단일 벡터 시스템을 나타낸 도면이다.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.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
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 .
실시예Example
실시예Example 1: 형질주입 시스템의 제작 1: Production of transfusion system
1.1. 벡터의 제작1.1. Production of vector
형질전환 동물(transgenic animal)을 제조하기 위한 형질주입 시스템(transfection system)을 제작하기 위하여, loxP-AQP3 shRNA를 발현하는 렌티바이러스(lentivirus) 및 HoxB7-Cre를 발현하는 렌티바이러스(lentivirus)를 제작하였다. 우선 렌티바이러스용 벡터를 제작하기 위하여, 마우스(mouse)의 아쿠아포린-3(aquaporin-3, AQP3) cDNA 서열을 기초로 하여, siRNA Selection Web Server(http://jura.wi.mit.edu/bioc/siRNA)에서 siRNA 타겟 사이트를 정하고, 대조군으로는 무작위 서열(scrambled sequence)을 사용하였고, 각각의 합성된 올리고뉴클레오티드(oligonucleotide)는 pSico lentiviral vector(Addgene)의 XhoⅠ-HpaⅠ 사이트에 삽입하였다. 염기서열(서열번호 1 내지 4)은 표 1에 나타내었다. 또한, HoxB7 프로모터(promoter)는 Puro-Cre empty vector(Addgene)의 XbaII-NheI 사이트에 삽입하였다. 각각의 제작된 벡터 디자인은 도 1에 나타내었다.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.
또한, 단일 벡터 시스템을 위해서는 "loxP-shAQP3" 벡터의 U6 프로모터 앞에 위치한 서열을 제한효소(XbaⅠ)로 자른 후, Hoxb7 프로모터-Cre recombinase 서열(서열번호 20)을 삽입하여 단일 벡터를 제작하였다. 제작된 단일 벡터 시스템 디자인은 도 6에 나타내었다.
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.
1.2. 형질전환된 1.2. Transformed 렌티바이러스Lentivirus 제작 making
인간의 배아 신장(human embryonic kidney, HEK) 293FT 세포주(Invitrogen)에 실시예 1.1의 방법으로 제조한 각각의 발현 벡터(expression vector)와 두 종류의 헬퍼 플라스미드(helper plasmid)인 pCMV △8.9 및 수포성 구내염 바이러스 G 단백질 플라스미드(vesicular stomatitis virus G protein plasmid)를 함께 칼슘-포스페이트 방법(calcium phosphate method)에 따라 형질주입(transfection)하였다. 그리고 72시간 후에 상층액을 수거하여 780g에서 5분간 원심분리하고, 0.45um 필터를 이용하여 걸러내고, 다시 83,000g에서 1.5시간 동안 원심분리를 시행하여 가라앉은 펠렛(pellet)을 수거하였다. 수거된 펠렛에 100uL의 PBS(phosphate buffered saline)를 첨가하고 렌티바이러스(lentivirus)의 양을 측정한 다음 4X108 transfection units/mL이 되도록 나누어 -80℃에서 사용시까지 보관하였다.
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.
실시예Example 2: 유전자 발현 억제 확인 2: Confirmation of gene expression inhibition
2.1. 2.1. inin vitrovitro 실험 Experiment
실시예 1의 방법으로 제작된 LV-Hoxb7 Cre 렌티바이러스와 LV-loxP shAQP3 렌티바이러스의 유전자 발현 억제능(gene silencing ability)을 확인하기 위하여, 집합관 세포(kidney collecting duct cell)와 혈관간막세포(mesangial cell)를 사용하였다. 각각의 세포에 렌티바이러스 4X105 TU를 처리하고, 48시간 배양한 후에 새로운 배양용 배지로 갈아준 후 다시 72시간 동안 추가 배양한 후에 배양된 세포를 수거하여 실험에 사용하였다. 형질주입 효율은 각각의 바이러스에 포함되어 있는 mCherry 및 EGFP의 발현량을 웨스턴 블로팅(western blotting)으로 확인하여 측정하였다. 웨스턴 블로팅은 수거된 세포를 SDS 시료용 완충용액(2% SDS, 10mM Tris-HCl, 10%(vol/vol) glycerol, pH6.8)을 이용하여 용해시킨 후, Laemmli 시료용 완충용액을 처리하고, 100℃에서 5분간 가열한 후에 12% acrylamide denaturing SDS-polyacrylamide 겔을 이용하여 전기영동하여 단백질을 분리한 후에, Hoeffer semidry blotting apparatus를 이용하여 Hybond-ECL 막(membrane)으로 이동시켰다. 단백질이 이동된 막은 blocking buffer A(1X PBS, 0.1% Tween 20, 5% nonfat milk)에 담가 실온에서 1시간 동안 반응시킨 후에 1:1,000으로 희석한 다클론성 항체(polyclonal antibody)인 AQP3(Abcam), EGFP(Abcam), mCherry(GeneFopoeia), 또는 beta-actin(Sigma-Aldrich)을 각각 처리한 후에 4℃에서 16시간 동안 반응시켰다. 반응이 종료된 막은 0.1% Tween 20이 첨가되어 있는 1X PBS를 이용하여 15분 동안 1회 세척하고, 5분간 2회 세척하여 결합되지 않은 항체를 제거하고, 1:2,000으로 희석된 horseradish peroxidase-linked donkey anti-goat IgG(Santa Cruz Biotechnology)가 포함되어 있는 blocking buffer A에서 다시 반응시킨 후에 다시 동일한 방법으로 세척하여 결합되지 않은 항체를 제거하였다. 세척된 막은 ECL 용액을 사용하여 현상(developing)하였다. 그리고 각각의 단백질 양은 TINA image software(Raytest)를 사용하여 측정하였다.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
또한, AQP3 RNA 양은 실시간 중합효소연쇄반응(real-time polymerase chain reaction)을 이용하여 측정하였다. RNA를 추출하기 위하여, 수거된 세포에 100uL의 RNA STAT-60 reagent(Tel-Test)를 첨가한 후에 냉동 및 해동 단계를 세 번을 반복하여 세포를 용해시킨 후에 700uL의 RNA STAT-60 reagent를 추가로 첨가한 후에 잘 섞은 후 5분간 실온에서 방치하였다. 그리고 160uL의 클로로포름(chloroform)을 첨가하고 30초 동안 강하게 섞어준 후에 12,000g, 4℃에서 15분간 원심분리하여 상층액만 수거하였다. 수거된 상층액에 400uL의 이소프로판올(isopropanol)을 첨가하고, 12,000g, 4℃에서 30분간 원심분리하여 RNA를 침전시키고, 70% 에탄올(ethanol)을 이용하여 세척한 후에 DEPC가 처리된 3차 증류수에 사용시까지 보관하였다. 추출된 RNA 2ug에 10uM random hexanucleotide primer, 1mM dNTP, 8mM의 MgCl2, 30mM KCl, 50mM Tris-HCl, 0.2mM dithithreithol, 25U RNase inhibitor, 및 40U AMV reverse transcriptase(Boehringer Mannheim cDNA synthesis kit)를 첨가한 후에 30℃에서 10분, 42℃에서 1시간 반응시키고, 99℃에서 5분간 가열하여 반응을 종료시켜 cDNA를 합성하였다. 그리고 합성된 cDNA를 이용하여 실시간 중합효소연쇄반응을 실시하였다. 실시간 중합효소연쇄반응에 사용된 프라이머 서열은 표 2에 나타내었다. 25ng의 cDNA(5uL)에 10uL SYBR Green PCR Master kit와 5pM의 프라이머 세트를 첨가하고, ABI PRISM 7700 S0equence Detection System(Applied Biosystems)을 이용하여 중합효소연쇄반응을 실시하였다. 중합효소연쇄반응은 95℃에서 9분간 초기 가열한 후에, 94.5℃에서 30초, 60℃에서 30초, 72℃에서 1분 순서로 35회 반복한 후에, 72℃에서 7분간 반응시켜 실시하였다. 그리고 60℃에서 95℃까지 분당 2℃로 증가되도록 온도를 설정하여, melting curve를 확인하고, comparative CT method를 이용하여 cDNA의 양을 측정하였다. 그 결과는 도 2에 나타내었다.
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.
도 2A에 나타난 바와 같이, LV-Hoxb7 Cre를 처리한 집합관 세포, LV-Hoxb7 Cre 및 LV-loxP scr을 처리한 집합관 세포에서 mCherry가 발현된 것을 확인할 수 있는 반면, EGFP는 LV-loxP shAQP3을 처리한 집합관 세포에서만 관찰되는 것을 확인하였다. 또한, 도 2B에 나타난 바와 같이, EGFP의 발현량과 관계없이 LV-Hoxb7 Cre 및 LV-loxP shAQP3을 함께 처리한 세포에서만 AQP3 유전자의 발현이 억제된 것을 확인하였다. 반면 도 2C에 나타난 바와 같이, 혈관간막세포에서는 mcherry가 발현되지 않으며, LV-Hoxb7 Cre를 처리한 세포 외에는 모두 EGFP를 발현하는 것을 확인하였다.
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. 2.2. inin vivovivo 실험(단일 형질주입) Experiments (single-trait injection)
실시예 1의 방법으로 제작된 LV-Hoxb7 Cre 렌티바이러스와 LV-loxP shAQP3 렌티바이러스의 유전자 발현 억제능(gene silencing ability)을 확인하기 위하여, 12마리의 수컷 C57BL/6J 마우스를 사용하였다. 24 내지 26g의 수컷 C57BL/6J 마우스를 Jackson laboratories에서 구입하고, 4개의 그룹으로 나누어 대조군으로 1mL의 PBS를 주입하고, 실험군으로는 4X108 TU의 LV-Hoxb7 Cre, LV-loxP shAQP3, 또는 LV-loxP scr을 각각 hydrodynamic tail vein injection 방식으로 0일, 4일, 및 8일에 형질주입(transfection)하였고, 각각의 마우스는 처음 형질주입한 날로부터 6주 후에 실험에 사용하였다. 형질주입 효율은 각각의 마우스의 간(liver), 비장(spleen), 신장(kidney), 기도(trachea), 및 결장(colon)을 추출하여 Semi-nested PCR 및 면역형광염색법(immunofluorescence staining)으로 확인하였다. Semi-nested PCR은 실시예 2.1과 동일한 방법으로 실시하였고, 사용한 각각의 프라이머 서열은 표 3에 나타내었다. 면역형광염색법을 위해서는 추출된 각각의 조직을 4um 높이 단위로 자른 후에 슬라이드(slide) 위에 올려놓고, 아세톤(acetone)에 담가 4℃에서 10분 동안 고정시키고, 10분간 건조시킨 후, 10% donkey serum에서 20분 동안 반응시키고, 1:1,000으로 희석된 EGFP 단클론항체(Abcam)을 처리하고 실온에서 3시간 동안 반응시켰다. 그리고 PBS로 세척하여 결합하지 않은 항체는 제거한 후에 Cy2(green)-conjugated anti-mouse IgG antibody(Research Diagnostics)를 처리하고 60분 동안 반응시킨 후에 1:500으로 희석된 단클론항체 rabbit anti-mCherry(Abcam) 또는 anti-AQP3(Abcam)을 처리하고 형광 현미경으로 확인하였다. 결과는 도 3에 나타내었다.
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.
도 3에 나타난 바와 같이, EGFP는 간, 비장 및 신장에서 모두 강하게 발현되는 반면, mCherry는 오직 신장에서만 발현되는 것을 확인하였다.
As shown in Fig. 3, EGFP was strongly expressed in liver, spleen and kidney, while mCherry was expressed only in kidney.
2.3. 2.3. inin vivovivo 실험(이중 형질주입) Experiment (double transfusion)
AQP3의 발현이 억제된 형질전환 동물이 정상적으로 제작되는지 확인하기 위하여, LV-Hoxb7 Cre, LV-loxP shAQP3, LV-Hoxb7 Cre 및 LV-loxP shAQP3을 각각 C57BL/6J 마우스에 형질주입하였다. 이중 형질주입을 위해서는, LV-HoxB7 Cre를 주입한 마지막 날 이후에 3일, 7일, 및 11일에 LV-loxP shAQP3를 주입하였다. 그리고 실시예 2.1과 동일한 방법으로 웨스턴 블로팅 및 실시간 중합효소연쇄반응을 실시하고, 실험 종료 하루 전에는 마우스의 소변을 24시간 동안 수거하여, 소변의 양(volume)을 측정하고, vapor pressure osmometer(5100C; Wescor)를 이용하여 삼투압(osmolality)를 측정하였다. 그 결과는 도 4 및 도 5에 나타내었다.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
도 4에 나타난 바와 같이, LV-Hoxb7 Cre를 형질주입한 마우스에서는 집합관 세포에서만 mCherry가 발현되고, LV-loxP shAQP3을 형질주입한 마우스에서는 집합관 세포를 포함한 모든 세포에서 EGFP 단백질의 발현이 증가되는 것을 확인하였다(도 4C). 그러나 두 실험군 모두 AQP3의 발현량, 소변량, 소변의 삼투압에는 변화가 없는 것을 확인하였다(도 4D). 반면, LV-Hoxb7 Cre 및 LV-loxP shAQP3을 형질주입한 마우스에서는 AQP3의 단백질 및 mRNA 발현량이 감소되었으며(도 4B), 소변량은 증가하고, 소변의 삼투압은 감소된 것을 확인하였다(도 4D).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).
또한, 도 5에 나타난 바와 같이, LV-Hoxb7 Cre 및 LV-loxP shAQP3을 형질주입한 마우스의 기도 및 결장에서 EGFP 단백질이 발현되지만, AQP3 단백질 및 mRNA의 발현량에는 변화가 없는 것을 확인하였다. 상기 결과를 통하여, Hoxb7 프로모터는 신장에서만 작동되며, LV-HoxB7 Cre 및 LV-loxP shAQP3 시스템이 신장에서 정상적으로 작동되는 것을 확인할 수 있었다.
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.
상기 결과들을 통하여, 본 발명의 형질전환 시스템은 in vitro 및 in vivo에서 모두 효과적으로 작동되는 것을 확인할 수 있었으며, 상기 형질전환 시스템을 이용하여 프로모터, 유전자 등을 원하는 종에 맞춰 전환하면 여러 동물의 성체에서 세포 특이적 유전자 발현 억제 형질전환 동물을 용이하게 제작할 수 있다는 것을 확인할 수 있었다.
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.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.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 tacctgtttt 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 <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)
(b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 형질주입하는 단계를 포함하고,
상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 목적 유전자의 발현을 억제하는 방법.(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 the expression of the target gene,
Wherein the second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences.
상기 세포특이적 프로모터는 서열번호 15의 염기서열을 포함하는 Hoxb7 프로모터인 것을 특징으로 하는, 방법.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.
상기 재조합 효소는 세포특이적 프로모터의 조절을 받는 것을 특징으로 하는, 방법.The method according to claim 1,
Wherein said recombinant enzyme is under the control of a cell-specific promoter.
상기 위치특이적 재조합 효소는 Cre 또는 Flp인 것을 특징으로 하는, 방법.The method according to claim 1,
Wherein the site-specific recombinase is Cre or Flp.
상기 위치특이적 재조합 효소가 인식하는 서열은 loxP 또는 FRT인 것을 특징으로 하는, 방법.The method according to claim 1,
Wherein the sequence recognized by the site-specific recombinase is loxP or FRT.
상기 목적 유전자의 발현 억제용 올리고뉴클레오티드는 목적 유전자의 shRNA(short hairpin RNA)인 것을 특징으로 하는, 방법.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.
상기 재조합 발현 벡터는 렌티바이러스(lentivirus), 아데노바이러스(adenovirus), 또는 레트로바이러스(retrovirus) 용인 것을 특징으로 하는, 방법.The method according to claim 1,
Wherein said recombinant expression vector is for lentivirus, adenovirus, or retrovirus.
(b) 목적하는 시기에 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 형질주입하는 단계를 포함하고,
상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 목적하는 시기의 목적 유전자의 기능을 확인하는 방법.(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,
Wherein said second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences.
상기 세포특이적 프로모터는 서열번호 15의 염기서열을 포함하는 Hoxb7 프로모터인 것을 특징으로 하는, 방법.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.
상기 재조합 효소는 세포특이적 프로모터의 조절을 받는 것을 특징으로 하는, 방법.9. The method of claim 8,
Wherein said recombinant enzyme is under the control of a cell-specific promoter.
상기 위치특이적 재조합 효소는 Cre 또는 Flp인 것을 특징으로 하는, 방법.9. The method of claim 8,
Wherein the site-specific recombinase is Cre or Flp.
상기 위치특이적 재조합 효소가 인식하는 서열은 loxP 또는 FRT인 것을 특징으로 하는, 방법.9. The method of claim 8,
Wherein the sequence recognized by the site-specific recombinase is loxP or FRT.
상기 목적 유전자의 발현 억제용 올리고뉴클레오티드는 목적 유전자의 shRNA(short hairpin RNA)인 것을 특징으로 하는, 방법.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.
상기 재조합 발현 벡터는 렌티바이러스(lentivirus), 아데노바이러스(adenovirus), 또는 레트로바이러스(retrovirus) 용인 것을 특징으로 하는, 방법.9. The method of claim 8,
Wherein said recombinant expression vector is for lentivirus, adenovirus, or retrovirus.
(b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 포함하고,
상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 목적 유전자의 발현 억제용 조성물.(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 suppressing expression of the target gene,
Wherein the second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences.
(b) 프로모터, 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 지역을 포함하는, 재조합 발현 벡터.(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.
(b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 인간을 제외한 동물에 형질주입하는 단계를 포함하고,
상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 인간을 제외한 형질전환동물의 제조 방법.(a) transfecting an animal other than a 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 the recognition sequence of the site-specific recombinase and an oligonucleotide for inhibiting expression of the target gene,
Wherein said second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences.
(b) 상기 위치특이적 재조합 효소의 인식 서열 및 목적 유전자의 발현 억제용 올리고뉴클레오티드(oligonucleotide)를 포함하는 제 2 재조합 발현 벡터를 포함하고,
상기 제 2 재조합 발현 벡터는 두 개의 재조합 효소 인식 서열 사이에 종결코돈(stop codon) 서열을 포함하는, 인간을 제외한 형질전환동물.(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 suppressing expression of the target gene,
Wherein said second recombinant expression vector comprises a stop codon sequence between two recombinant enzyme recognition sequences.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR102070763B1 (en) * | 2018-08-21 | 2020-01-29 | 성균관대학교산학협력단 | Dopaminergic neuron specific expression control system |
| KR20210058098A (en) * | 2019-11-13 | 2021-05-24 | 주식회사 마크로젠 | Neuron-specific promoter and recombinant vector comprising the same for inducing DNA modification |
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| KOHAN, DE., ‘Progress in gene targeting: using mutant mice to study renal function and disease’, Kidney International, 2008, 74권, 427-437면 (2008.4.16. 온라인 공개). * |
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| KR102070763B1 (en) * | 2018-08-21 | 2020-01-29 | 성균관대학교산학협력단 | Dopaminergic neuron specific expression control system |
| KR20210058098A (en) * | 2019-11-13 | 2021-05-24 | 주식회사 마크로젠 | Neuron-specific promoter and recombinant vector comprising the same for inducing DNA modification |
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