KR101444297B1 - CYP4F16 knock-down mouse and manufacturing method thereof - Google Patents
CYP4F16 knock-down mouse and manufacturing method thereof Download PDFInfo
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Abstract
본 발명은 CYP4F16 유전자가 넉다운된 형질전환마우스 및 그 제조방법에 관한 것이다. 보다 상세하게는, 수면병에 대한 저항성을 나타내는 CYP4F16유전자가 넉다운된 형질전환마우스 및 그 제조방법에 관한 것이다. 본 발명의 형질전환 마우스를 이용하면, 트리파노솜의 체내 침입후 발생되는 면역관련 유전자 중 수면병의 발생생원인 규명하고, 이를 치료할 수 있는 신약을 개발하는 데 이용될 수 있다.The present invention relates to a transgenic mouse knocked down with a CYP4F16 gene and a method for producing the same. More particularly, the present invention relates to transgenic mice knocked down by the CYP4F16 gene showing resistance to sleeping sickness, and a method for producing the same. The use of the transgenic mouse of the present invention can be used to develop a new drug that can identify the origin of sleeping sickness among the immune-related genes generated after invasion of the body of tripopanos and to treat them.
Description
본 발명은 CYP4F16 전자가 넉다운된 형질전환마우스 및 그 제조방법에 관한 것이다. 보다 상세하게는, 수면병에 대한 저항성을 나타내는 CYP4F16유전자가 넉다운된 형질전환마우스 및 그 제조방법에 관한 것이다.
The present invention relates to CYP4F16 electron knockdown transgenic mice and methods for their production. More particularly, the present invention relates to transgenic mice knocked down by the CYP4F16 gene showing resistance to sleeping sickness, and a method for producing the same.
인간 아프리카 트리파노소마증은 기생충 매개체 질환이다. 관련 기생충은 트리파노소마 (Trypanosoma) 속에 속하는 원생동물이다.Human African trypanosomiasis is a parasitic disease. Related parasites are protozoa belonging to the genus Trypanosoma.
수면병으로도 알려져 있는 이 병은 원생동물의 편모충류의 일종인 감비아트리파노소마(Trypanosoma gambiense) 또는 로데시아트리파노소마(T. rhodesiense)의 감염으로 일어나는 병이다.This disease, also known as sleeping sickness, is caused by infection of the protaminant Trypanosoma gambiense or T. rhodesiense, a protozoan species of protozoa.
이들은 인간 병원성 기생충이 잠복해 있는 인간 또는 동물로부터 감염원을 획득한 체체 파리 (글로시나 (Glossina) 속)에 물림으로써 인간에게 전파된다.They are transmitted to humans by binding to a flies (Glossina) that have acquired infections from humans or animals lurking in human pathogenic parasites.
수면병에 항기생충제로서 평가하고 사용하기 위한 신규 화합물의 개발이 바람직하며, 이러한 약물 개발을 위해 인수공통 전염병인 수면병에 대한 모델 동물 생산이 필요한 시점이다.The development of new compounds for evaluation and use as antiparasitic agents against sleeping sickness is desirable, and it is time to develop model animals for sleeping sickness, a common infectious disease, for the development of these drugs.
이러한 종류의 모델 동물에 대한 선행기술로는, 퇴행성 신경계 질환에 대한 저항성을 가지는 ＣＩＩＡ 형질전환 마우스가 한국등록특허 1055376에 공개되어 있다.
As a prior art for this type of model animal, a CIIA transgenic mouse having resistance to degenerative nervous system diseases is disclosed in Korean Patent No. 1055376.
본 발명자들은 수면병에 관한 연구 및 이러한 질환에 대한 치료제를 개발할 수 있는 동물모델을 제작하고자 연구한 결과, 본 발명을 완성하기에 이르렀다.The present inventors have completed the present invention by studying sleeping sickness and producing animal models capable of developing therapeutic agents for such diseases.
결국, 본 발명의 목적은 CYP4F16 유전자가 넉다운된 형질전환마우스 및 그 제조방법을 제공하는 것이다. Accordingly, it is an object of the present invention to provide a transgenic mouse knocked down with CYP4F16 gene and a method for producing the same.
본 발명의 또 다른 목적은 상기 형질전환 마우스의 제조방법을 제공하는 것이다.
It is still another object of the present invention to provide a method for producing the transgenic mouse.
본 발명의 일 측면에 따르면, 프로모터 및 이와 작동가능하게 연결된 서열번호 1로 표시되는 마우스 CYP4F16 넉다운용 siRNA 발현 재조합 벡터가 제공될 수 있다.According to one aspect of the present invention, a promoter and a siRNA expression recombinant vector for mouse CYP4F16 knockdown represented by SEQ ID NO: 1 operably linked thereto may be provided.
일 실시예에 따르면, 상기 프로모터는 U6 프로모터일 수 있다.According to one embodiment, the promoter may be a U6 promoter.
본 발명의 다른 측면에 따르면, 상기 재조합 벡터가 도입된 형질전환 마우스가 제공될 수 있다.According to another aspect of the present invention, a transgenic mouse into which the recombinant vector has been introduced may be provided.
본 발명의 또 다른 측면에 따르면, (a) 프로모터 및 이와 작동가능 하게 연결된 서열번호 1로 표시되는 CYP4F16 발현 억제용 siRNA 발현카세트를 제조하는 단계; (b) 상기 재조합 융합 유전자를 마우스의 수정란에 도입하는 단계; 및 (c) 상기 수정란을 대리모 마우스에 착상시켜 새끼를 생산하는 단계를 포함하는 CYP4F16 유전자 발현이 넉다운된 형질전환 마우스의 제조방법이 제공될 수 있다.
According to a further aspect of the present invention there is provided a method for producing a siRNA expression cassette comprising: (a) preparing a promoter and siRNA expression cassette for inhibiting expression of CYP4F16 expressed by SEQ ID NO: 1 operatively linked thereto; (b) introducing the recombinant fusion gene into a mouse embryo; And (c) fusing the embryo to a surrogate mouse to produce a cub. The method of producing a transgenic mouse knocked down by CYP4F16 gene expression can be provided.
본 발명의 형질전환 마우스를 이용하면, 트리파노솜의 체내 침입후 발생되는 면역관련 유전자 중 수면병의 발생원인이 되는 유전자를 규명하고, 이를 치료할 수 있는 신약을 개발하는 데 이용될 수 있다.
Use of the transgenic mouse of the present invention can be used to identify a gene responsible for sleeping sickness among immune-related genes generated after invasion of the body of tripopanos and to develop a new drug that can treat the gene.
도 1은 siRNA 방법을 이용하여 CYP4F16 유전자를 넉다운시키기 위한 벡터를 나타낸 도면이다.
도 2는 Cyp4f16 shRNA 넉다운 과정을 나타내는 흐름도이다.
도 3은 미세주입을 위해 제조한 Cyp4f16-shRNA 단편을 나타낸다.
도 4는 shRNA-CYP4F16 (A/J) 형질전환마우스를 검색한 전기영동결과를 나타낸다.
도 5는 본 발명에 따라 제조된shRNA-CYP4F16 (A/J) 형질전환마우스이다.
도 6은 shRNA-CYP4F16 (C57BL/6) 형질전환마우스를 검색한 전기영동결과를 나타낸다.
도 7은 shRNA-CYP4F16 (C57BL/6) 형질전환마우스이다.
도 8은 다양한 조직에서의 cyp4f16 전사물에 대한 Real-time RT-PCR 결과를 나타낸다.
도 9는 비장에서의 mCYP4F16 (C57BL) mRNA 발현에 대한 것이다.
도 10은 F1(A/J) 에서 F0 42(♀)에 대한 Tg 스크리닝 결과를 나타낸다.
도 11은 형질전환마우스의 췌장(pancreas)의 HE 염색결과이다.
도 12는 형질전환마우스의 비장(spleen)의 HE 염색결과이다.
도 13은 형질전환마우스의 정소(testis)의 HE 염색결과이다.Brief Description of the Drawings Figure 1 shows a vector for knocking down the CYP4F16 gene using the siRNA method.
Figure 2 is a flow chart illustrating the Cyp4f16 shRNA knockdown procedure.
Figure 3 shows a Cyp4f16-shRNA fragment prepared for microinjection.
Fig. 4 shows electrophoresis results of shRNA-CYP4F16 (A / J) transgenic mice.
Figure 5 is an shRNA-CYP4F16 (A / J) transgenic mouse prepared according to the present invention.
Fig. 6 shows electrophoresis results of shRNA-CYP4F16 (C57BL / 6) transgenic mice.
Figure 7 is an shRNA-CYP4F16 (C57BL / 6) transgenic mouse.
Figure 8 shows Real-time RT-PCR results for cyp4f16 transcripts in various tissues.
Figure 9 is for mCYP4F16 (C57BL) mRNA expression in spleen.
Figure 10 shows the Tg screening results for F1 (A / J) to F042 ().
Fig. 11 shows HE staining results of pancreas of transgenic mice.
Figure 12 shows the result of HE staining of the spleen of transgenic mice.
Fig. 13 shows HE staining results of testis of transgenic mice.
본 발명은 CYP4F16 유전자가 넉다운된 형질전환마우스 및 그 제조방법에 관한 것이다. 보다 상세하게는, 수면병에 대한 저항성을 나타내는 CYP4F16유전자가 넉다운된 형질전환마우스 및 그 제조방법에 관한 것이다.
The present invention relates to a transgenic mouse knocked down with a CYP4F16 gene and a method for producing the same. More particularly, the present invention relates to transgenic mice knocked down by the CYP4F16 gene showing resistance to sleeping sickness, and a method for producing the same.
연구결과를 요약하면 다음과 같다.The results of the study are summarized as follows.
1. shRNA-CYP4F16 유전자가 넉다운된 마우스를 생산하기 위해 넉다운 DNA 벡터를 제작하여 단일세포(one cell) 수정란에 미세주입하여 대리모에 이식하였다. 그 결과 A/J 마우스 strain으로부터 shRNA-CYP4F16 transgene를 가지고 있는 암컷 1, 수컷 1두의 마우스를 생산하였다1. A knockdown DNA vector was constructed to produce a knockdown mouse shRNA-CYP4F16 gene, which was microinjected into a single cell embryo and transplanted into a surrogate mother. As a result, mice of 1 female and 1 male having shRNA-CYP4F16 transgene were produced from A / J mouse strains
2. 또한 DNA 미세주입 방법으로 C57BL/6N 마우스 strain으로 50 마리의 산자가 생산되었으며 그 중 수컷 3마리 그리고 암컷 3마리 총 6마리가 shRNA-CYP4F16 transgene를 가지고 있는 것으로 확인되었다. In addition, 50 strains of C57BL / 6N mouse strains were produced by DNA microinjection method, and 3 of 3 males and 3 of 3 females were found to have shRNA-CYP4F16 transgene.
3. 비장(Speen)에서 CYP4F16 mRNA 발현량을 조사하였을 때, 대조구에서 개체마다 변이가 심하였고, 이것은 또한 CYP4F16 transgene들에서도 마찬가지인 것으로 나타났으나. 형질전환마우스는 유의적으로 발현이 낮은 것으로 나타나 대조구와 차이를 보였다. 따라서 이 동물 및 세포주를 모델로 이용하여 수면병과 관련된 연구에 이용할 수 있을 것이다.
3. When CYP4F16 mRNA expression levels in Speen were examined, the variation in each individual was severe in the control, which was also the same in the CYP4F16 transgene. Transgenic mice were significantly lower in expression than control. Therefore, this animal and cell line can be used as a model for research related to sleeping sickness.
이하, 본 발명의 CYP4F16 유전자가 넉다운된 형질전환 마우스의 제작방법을 단계별로 설명하기로 한다. Hereinafter, the method for producing a transgenic mouse knocked down by the CYP4F16 gene of the present invention will be described step by step.
CYP4F16 유전자를 코딩하는 뉴클레오타이드 서열을 갖는 핵산은 siRNA가 유도되도록 재조합 벡터에 삽입된 다음, 마우스에 형질전환된다. A nucleic acid having a nucleotide sequence encoding a CYP4F16 gene is inserted into a recombinant vector such that siRNA is induced, and then transformed into a mouse.
본 발명에서 "넉다운"은 유전자의 발현을 완전히 차단시키는 "넉-아웃"과는 달리, 유전자의 발현양을 상당량 줄여 유전자의 기능이 감소되도록 하는 것을 의미한다. 이는 유전자의 전사체 수준에서 조절될 수도 있고, 단백질 수준에서 조절될 수도 있다.In the present invention, "knockdown" means that the function of the gene is reduced by significantly reducing the expression amount of the gene, unlike the " knock-out " It can be regulated at the transcript level or at the protein level of the gene.
본 발명에서 “벡터(vector)”란 목적 단백질을 코딩하는 핵산 서열을 숙주 세포로 도입하기 위한 수단을 의미한다. 적합한 벡터는 프로모터,오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 인핸서 같은 발현 조절 요소 외에도 막 표적화 또는 분비를 위한 신호 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있는데, 본 발명의 벡터는 넉다운시키고자 하는 유전자의 siRNA가 생성될 수 있도록 전사개시 신호와 프로모터가 포함된 것이 특징이다.&Quot; Vector " in the present invention means a means for introducing a nucleic acid sequence encoding a target protein into a host cell. Suitable vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal, enhancer, and may be prepared in various ways depending on the purpose, Is characterized by including a transcription initiation signal and a promoter so that siRNA of the gene to be knocked down can be generated.
상기 벡터의 프로모터는 구성적 또는 유도성일 수 있으며, 본 발명의 효과를 위해 추가적으로 변형될 수 있다. 본 발명의 일 실시예에서는 pU6 프로모터를 이용한다. 또한 발현벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택성 마커를 포함하고, 복제 가능한 발현벡터인 경우 복제 기원(Ori)을 포함한다. 벡터는 자가 복제하거나 숙주 게놈 DNA에 통합될 수 있다.
The promoter of the vector may be constitutive or inducible and may be further modified for the purposes of the present invention. In one embodiment of the present invention, the pU6 promoter is used. The expression vector also includes a selectable marker for selecting a host cell containing the vector, and a replication origin (Ori) in the case of a replicable expression vector. The vector may be self-replicating or integrated into the host genomic DNA.
바람직하게는 벡터 내로 삽입되어 전달된 유전자가 숙주세포의 게놈 내로 비가역적으로 융합되어 세포 내에서 유전자 발현이 장기간 안정적으로 지속되도록 하는 것이 바람직하다. Preferably, the gene is inserted into the vector so that the transferred gene is irreversibly fused into the genome of the host cell, so that the gene expression in the cell can be stably maintained for a long period of time.
구체적으로, 본 발명의 형질전환 마우스 제조에 사용된 벡터는, pU6shX 벡터(VectorCoreA, 대한민국)이다.Specifically, the vector used in the production of the transgenic mouse of the present invention is pU6shX vector (VectorCoreA, Korea).
도 2에는 siRNA 이중가닥이 형성되게 하기 위하여 필요한 삽입용 DNA 단편을 제조한 흐름도가 나타나 있다. 본 발명의 벡터는 숙주 게놈으로 삽입되어(integrate) 안정하게 유지됨을 특징으로 한다.
FIG. 2 shows a flow chart for preparing a DNA fragment for insertion required for forming a siRNA double strand. The vector of the present invention is characterized by being integrated into the host genome and stably maintained.
본 발명은 CYP4F16 유전자가 넉다운된 형질전환 마우스에 관한 것으로 본 발명에서 용어, “형질전환 (transgenic, Tg)"이란 세포 내 CYP4F16 유전자 전사체의 수준이 정상 세포 수준에 비하여 감소되도록 형질의 변형이 유도된 동물을 의미한다.The term "transgenic (Tg)" as used herein refers to a transgenic mouse knocked down by the CYP4F16 gene. In the present invention, the term "transgenic (Tg)" refers to a gene that induces a change in the trait such that the level of intracellular CYP4F16 gene transcript is reduced Lt; / RTI >
본 발명에서 “동물 모델(animal model)” 또는 “질환 모델(disease model)"은 사람의 질병과 유사한 특정 질환을 가지고 있어, 병인을 규명하고 병태를 확인할 수 있는 연구 대상이 될 수 있는 모델이 되는 동물을 의미한다. 동물 모델로서 사용하기 위한 동물은, 인간에서와 같은 효과를 예측할 수 있으며, 쉽게 만들 수 있고, 재현성이 있다. 또한, 인간질병의 병인과 같거나 유사하게 진행되어야 한다. 따라서, 인간과 같은 포유류 척추동물이면서, 장기 등의 체내 구조, 면역체계, 체온 등이 유사하고, 고혈압, 암, 면역결핍 등의 질환을 앓는 동물이 동물 모델로서 적합하다. 이런 동물은 바람직하게는 말, 양, 돼지, 염소, 낙타, 영양, 개, 래빗, 마우스, 래트, 기니피그, 햄스터 등의 포유류이고, 보다 바람직하게는 마우스, 래트, 기니피그, 햄스터 등의 설치류이다. 특히, 마우스는 소형동물로 번식력이 우세하고, 사양관리가 쉽고, 질병에 강하며, 유전적으로 균일하며, 다양한 종류가 개발되었고, 사람에서 발생하는 질병과 같거나 유사한 증상을 보이는 동물의 생산이 가능하여, 인간의 질병을 연구하는데 가장 많이 이용되고 있다.
The term " animal model " or " disease model "in the present invention refers to a disease model having a specific disease similar to a human disease, An animal for use as an animal model can predict the same effects as in humans, can easily be made, is reproducible, and must proceed in a manner similar to or similar to the etiology of human disease. Animal models such as mammalian vertebrate animals such as humans and animals with similar organs such as organs, immune system, body temperature and the like, and diseases such as hypertension, cancer, immunodeficiency, etc. are suitable as animal models. Mammals such as rabbits, sheep, pigs, goats, camels, nutrition, dogs, rabbits, mice, rats, guinea pigs and hamsters are preferred, and rodents such as mice, rats, guinea pigs and hamsters In particular, the mouse is a small animal that is predominantly fertile, is easy to manage specifications, is disease-resistant, genetically homogeneous, has developed a variety of species, and produces animals with similar or similar symptoms It is most often used to study human diseases.
상기의 CYP4F16 유전자가 넉다운된 형질전환 마우스는 트리파노소마 관련 질환의 치료, 예방, 진단 또는 그 분자적 기전에 관한 연구과정 등에 널리 이용될 수 있다.The CYP4F16 gene knockdown transgenic mouse can be widely used for the treatment, prevention, diagnosis of a disease associated with trypanosomiasis, or a study on its molecular mechanism.
상기 기술된 바와 같이 본 발명은 CYP4F16 유전자가 넉다운된 형질전환 마우스의 제작방법을 제공한다. 본 발명의 CYP4F16 유전자가 넉다운된 형질전환 마우스는 트리파노소마 관련 질환인 수면병의 연구과정 및 치료제 개발에 널리 활용될 수 있을 것이다.
As described above, the present invention provides a method for producing a transgenic mouse knocked down by the CYP4F16 gene. The CYP4F16 gene knockdown transgenic mouse of the present invention can be widely used for research and development of therapeutic agents for sleep apnea, which is a disease caused by Trypanosoma.
이하, 본 발명을 보다 구체적으로 설명하기 위하여 하기 실시예 등을 들어 설명한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며 본 발명의 범위가 아래에서 상술하는 실시예들에 한정되는 것으로 해석돼서는 안 된다. 본 발명의 실시예들은 본 발명의 구체적 이해를 돕기 위해 예시적으로 제공되는 것이다.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided by way of example to facilitate a specific understanding of the present invention.
실시예Example 1. One. CYP4F16CYP4F16 유전자 넉다운용 벡터의 제조 Generation of vectors for gene knockdown
도 2에는 CYP4F16 유전자 넉다운용 벡터의 제조과정이 나타나 있다.FIG. 2 shows a preparation process of a vector for CYP4F16 gene knockdown.
세부과정을 보다 상세하게 설명하면 다음과 같다.The detailed procedure will be described in more detail as follows.
A. 마우스 Cyp4f16 (GI:13277361) 로 부터 shRNA 넉다운에 유리한 염기서열을 선정A. Selection of a base sequence favorable for shRNA knockdown from mouse Cyp4f16 (GI: 13277361)
B. 벡터에 유전자를 삽입하기 위하여 EcoRI 및 XhoI 제한효소 사이트를 포함하는 Cyp4f16 유전자 단편.B. Cyp4f16 gene fragment containing EcoRI and XhoI restriction enzyme sites to insert genes into vectors.
C. 완성된 벡터의 구조C. Structure of the Completed Vector
D. RNA U6 중합효소에 의해 생성된 shRNAD. RNA RNA produced by U6 polymerase
E. RNAi 메카니즘에 따른 Cyp4f16 mRNA의 넉다운
E. Knockdown of Cyp4f16 mRNA by RNAi mechanism
2) Cyp4f16-shRNA fragment를 준비하기 위해 DNA microinjection 방법을 활용하여 Cyp4f16 유전자의 발현이 knock-down된 transgenic 생쥐를 생산하기 위해 pU6shX-mCYP4F16로부터 PvuII 제한효소로 처리하였을 때 U6 RNA polymerase promotor와 Cyp4f16-shRNA sequence가 포함된 783 bp fragment 를 넣었고 이들을 agarose로부터 분리/정제하여 형질전환 생쥐를 생산하는데 활용하였다 (도 3).
2) To prepare transgenic mice knocked down by the expression of Cyp4f16 gene using DNA microinjection method to prepare Cyp4f16-shRNA fragment, U6 RNA polymerase promoter and Cyp4f16-shRNA were treated with PvuII restriction enzyme from pU6shX-mCYP4F16 sequence, and these fragments were separated / purified from agarose and used to produce transgenic mice (FIG. 3).
실시예Example 2. 넉다운 마우스의 생산 및 검정 2. Production and assay of knockdown mice
1) shRNA-CYP4F16이 넉다운된 마우스를 생산하기 위해 상기 실시예 1에 따라 제조된 넉다운 DNA 벡터를 제작하여 단일세포 수정란에 미세주입 하여 대리모에 이식하였다. 그 결과 A/J 마우스 strain으로부터 25 마리의 산자가 생산되었으며 A/J 마우스 strain로부터 F0#24(A/J)가 shRNA-CYP4F16 transgene를 가지고 있는 것으로 확인되었고, 암컷 1, 수컷 1두가 각각 생산되었다 (도 4, 5). 1) Knockdown DNA vector prepared according to Example 1 was prepared to produce shRNA-CYP4F16 knockdown mouse, and microinjected into single cell embryos and transplanted into surrogate mothers. As a result, 25 strains were produced from A / J mouse strains and F0 # 24 (A / J) from A / J mouse strains was found to have shRNA-CYP4F16 transgene. One female and one male produced (Figs. 4 and 5).
2) 또한 DNA 미세주입 방법으로 C57BL/6N 마우스 strain으로 50 마리의 산자가 생산되었으며 수컷 3마리 그리고 암컷 3마리 총 6마리가 shRNA-CYP4F16 transgene을 가지고 있는 것으로 확인되었다. 이들 중 F0#3(C57BL/6)는 수컷이며 2010년 9월 8일에 생산, F0#14(C57BL/6)는 수컷이며 2010년 9월 12일에 생산, F0#24(C57BL/6)는 암컷이며 2010년 9월 12일에 생산, F0#28(C57BL/6)는 암컷이며 2010년 9월 14일에 생산, F0#34(C57BL/6)는 암컷이며 2010년 9월 14일에 생산, 그리고 F0#40(C57BL/6)는 수컷이며 2010년 9월 14일에 생산되었다 (도 6, 7). 2) In addition, 50 strains of C57BL / 6N mouse strains were produced by DNA microinjection method and 3 shrimp and 3 females totaled 6 shRNA-CYP4F16 transgene. Among them, F0 # 3 (C57BL / 6) is male and produced on September 8, 2010. F0 # 14 (C57BL / 6) is male and produced on September 12, Is a female, produced on September 12, 2010, F0 # 28 (C57BL / 6) is female and produced on September 14, 2010, F0 # 34 (C57BL / 6) is female and on September 14, 2010 Production and F0 # 40 (C57BL / 6) were males and were produced on September 14, 2010 (Figures 6 and 7).
3) DNA 미세주입 방법으로 생산된 shRNA-CYP4F16 넉다운 마우스에서 도 8과 같이 각 조직별 CYP4F16 유전자의 넉다운을 확인하였다. 3) In the shRNA-CYP4F16 knockdown mouse produced by the DNA microinjection method, the knockdown of the CYP4F16 gene in each tissue was confirmed as shown in FIG.
4) 비장(Speen)에서 CYP4F16 mRNA 발현량을 조사하였을 때, 대조구에서 개체마다 변이가 심하였고 (16~918), 이것은 또한 CYP4F16 transgene들에서도 마찬가지인 것으로 나타났으나(1~830). 하지만, TG53, 61, 그리고 74는 유의적으로 발현이 낮은 것으로 나타나 대조구와 차이를 보였다. 대조구에서 이상이 있는 것으로 보이는 4번과 8번 마우스는 제외하였다. 흥미로운 것은 형질전환마우스에서 이상이 있는 52번과 72번 마우스는 다른 형질전환마우스에 비해 매우 높은 cyp4f16 발현을 보였다.
4) The expression level of CYP4F16 mRNA in Speen showed that the control was highly variable (16 ~ 918), which was also the same in CYP4F16 transgene (1 ~ 830). However, TG53, 61, and 74 were significantly lower in expression than control. Except for
실시예Example 3. 넉다운 마우스 후대 생산 및 특성 3. Generation and Characteristics of Knockdown Mouse
1) shRNA-CYP4F16 유전자가 넉다운된 마우스를 일반 마우스와 교배하여 F1을 생산하였고 F1에 유전자가 전이되었는지를 확인한 결과 도 9 및 도 10의 결과와 같이 모두 7마리의 후대 중 2마리(#2, 4번)에서 전이된 유전자가 발견되어 형질전환된 후대를 생산하여 연구에 제공할 수 있음을 확인하였다.1) Mice knocked down with shRNA-CYP4F16 gene were crossed with normal mice to produce F1. As a result of checking whether the gene was transferred to F1, 2 out of 7 mice (# 2, 4), and found that transgenic transgenes could be produced and provided for research.
2) shRNA-CYP4F16 유전자가 넉다운된 마우스의 체중 및 비장 중량(spleen weight) 등 특성은 표 1과 같다.
2) Weight and spleen weight of mice knocked down by shRNA-CYP4F16 gene are shown in Table 1.
편차 Deviation
표 1을 참조하면, 형질전환마우스의 체중 대비 비장중량이 대조구에 비해 낮은 것을 알 수 있다.Referring to Table 1, it can be seen that the spleen weight of the transgenic mice is lower than that of the control.
도 11 내지 도 14에는 상기에서 제조된 넉다운 마우스의 췌장, 비장 및 정소에 대한 HE 염색 사진이 나타나 있다. 하기의 사진을 참고하면, 본 발명에 따른 형질전환마우스가 나타내는 조직해부학적 이상을 관찰할 수 있다.FIGS. 11 to 14 show HE staining photographs of the pancreas, spleen and testes of the knockdown mice prepared above. The tissue anatomical anomaly exhibited by the transgenic mouse according to the present invention can be observed by referring to the following photographs.
도 11의 경우, 췌장(pancreas)조직을 HE(헤마톡실린 & 에오신) 염색하여 200 배 배율로 관찰한 것이다. 도 10을 참조하면, 소엽사이의 관(interlobular duct) 주변에서 염증성 세포의 인입(infiltration)이 관찰되었다.In the case of Fig. 11, the pancreas tissue was stained with HE (hematoxylin and eosin) and observed at a magnification of 200 times. Referring to FIG. 10, infiltration of inflammatory cells was observed around the interlobular duct.
도 12의 경우, 비장(spleen) 조직을 HE 염색하여 400배 배율로 관찰한 것이다. 도 11을 참조하면, 황색 색소(yellow pigment)의 침전이 관찰됨을 알 수 있다(화살표시)In the case of Fig. 12, the spleen tissue was HE-stained and observed at a magnification of 400 times. Referring to FIG. 11, it can be seen that precipitation of a yellow pigment is observed (at the time of an arrow)
도 13의 경우, 정소(testis) 조직을 HE 염색하여 400배 배율로 관찰한 것이다. 도 13을 참조하면, (A)에서는 다핵의 거대세포가 관찰되었으며, (B)에서는 도관의 위축(tubular atrophy)이 관찰되었다. (C)에서는 정자발생 단계 IX-X기의 세정관(seminiferous tubule) 내 정자유리(spermiation)가 저해되고 있음이 관찰되었다.
In the case of FIG. 13, the testis tissue was HE-stained and observed at a magnification of 400 times. Referring to FIG. 13, multinucleated giant cells were observed in (A), and tubular atrophy was observed in (B). (C), spermiation in the seminiferous tubule of spermatogenesis stage IX-X was inhibited.
본 발명의 형질전환 마우스를 이용하면, 트리파노솜의 체내 침입후 발생되는 면역관련 유전자 중 수면병의 발생생원인 규명하고, 이를 치료할 수 있는 신약을 개발하는 데 이용될 수 있다.
The use of the transgenic mouse of the present invention can be used to develop a new drug that can identify the origin of sleeping sickness among the immune-related genes generated after invasion of the body of tripopanos and to treat them.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항 들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
SEQUENCE LISTING <110> RURAL DEVELOPMENT ADMINISTRATION <120> CYP4F16 knock-down mouse and manufacturing method thereof <130> NPF21076 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 49 <212> DNA <213> Mus musculus <400> 1 tcatggcatc ttctgctgct ttcaagagaa gcagcagaag atgccatga 49 SEQUENCE LISTING <110> RURAL DEVELOPMENT ADMINISTRATION <120> CYP4F16 knock-down mouse and manufacturing method thereof <130> NPF21076 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 49 <212> DNA <213> Mus musculus <400> 1 tcatggcatc ttctgctgct ttcaagagaa gcagcagaag atgccatga 49
Claims (4)
A siRNA-expressing recombinant vector for knockdown of the CYP4F16 gene comprising the nucleotide sequence of SEQ ID NO: 1 operably linked to the U6 promoter.
A transgenic mouse into which the recombinant vector of claim 1 has been introduced.
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