저자는 (학과 안과 및 Visual 과학)과 의학의 세인트 루이스 학교 워싱턴 대학에서 마우스 유전학 코어의 분자 유전학 코어, 형태 및 이미지 코어의 직원을 감사드립니다. 저자는 세포에 대한 맥도넬 센터 및 분자 신경 생물학, 신경 장애에 대한 희망 센터에서 작은 보조금 프로그램에 의해 지원, 국립 안과 연구소 (DH에 # R01EY022632), 국립 안과 연구소 센터 코어 그랜트 (# 1 P30EY002687) 및 연구에서 무제한 부여 안과 및 Visual 과학 부서에 실명을 방지합니다.

윤리 문 : 동물 주제와 관련된 절차는 세인트 루이스의 워싱턴 대학에서 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1. 마우스 사육 및 유전자형 <ol><li> (PCP2Cre CAG-EGFP / KASH2) 19, 20, Tg는 생산의 Tg (CAG-LacZ를 / EGFP-KASH2)의 Tg와 마우스 (PCP2-Cre 호텔) 쥐 새끼를 낳. 주 :이 프로토콜의 나머지는 소뇌 내 조롱박 세포 EGFP-KASH2 발현을 제한의 Tg (PCP2-Cre 호텔) 마우스 라인의 사용에 초점을 맞추고 있지만, 유사한 절차가 다른 Tg는 (Cre 호텔) 마우스 수 미행 ( 도 2A). </li><li> 이전 19 설명 된대로 모두 EGFP와 Cre 호텔 유전자를 품고 새끼를 식별하는 유전자형을 수행합니다. </li></ol> 해부 및 조직 컬렉션 재료 2. 준비 <ol><li> -20 ℃에서 30 %의 수크로오스 PBS (PBS 1X 중 자당 30g / 100 mL) 및 점포 100 ㎖를 준비한다. 조직 (H)의 일transcardial 관류에 대한 35 ML의 주사기에 arvesting, 해동 및 자당 용액의 부하 ~ 30 ~ 35 ml의. </li><li> PBS로 4 % PFA, 40 ㎖ (10 ㎖, 16 % 파라 포름 알데히드 및​​ PBS 30 ㎖)을 준비한다. transcardial 관류에 대한 35 ML의 주사기에로드 ~ 30 ~ 35 ml의 4 % PFA. </li><li> 조직의 절개에 대한 PBS의 10 ㎖로 10cm 조직 배양 판을 채 웁니다. </li><li> 16 ㎎ / ㎖ 케타민 3 ㎎ / ㎖ 자일 라진 구성된 케타민 / 자일 라진 칵테일을 준비합니다. </li><li> 해부에 대한 70 % 에탄올 용액을 준비합니다. </li></ol> 3. 해부 및 조직 컬렉션 <ol><li> 1 ML의 주사기를 사용하여, 체중 25g 당 케타민 / 자일 라진 칵테일 500 μl를 주입하고 15 분 ~ 기다립니다. 대안 적으로, 관심있는 생물학적 시스템에 적합한 다른 제도적 승인 진정 방법을 사용한다. </li><li> 진정되면, 부드럽게의 등쪽면 스티로폼 트레이에 모든 발 핀, 통증 반응의 부족을 확인하기 위해 후면 발을 꼬집어마우스를 아래로 향하게. </li><li> 70 % 에탄올로 복부를 스프레이하고 마음에 액세스 할 흉곽의 상단에 하복부에서 절개를합니다. </li><li> 심장의 우심방의 작은 절개를 확인하고 심장의 좌심실에 30 % 자당 용액이 들어있는 주사기를 삽입합니다. </li><li> ~ 2-5 ml의 당 분의 속도로 용액을 관류. 비어있는 경우, 주사기를 제거하고 4 % PFA 용액으로 반복한다. </li><li> 스티로폼 트레이에서 마우스를 고정 해제 뇌에 액세스하기 위해 목을 벨. </li><li> 집게와 가위를 사용하여 동물의 머리 주위 피부를 제거합니다. 바늘로 두개골의 뒤쪽 부분에 구멍을 찌를 조심스럽게 가위 해부 집게와 두개골을 제거합니다. </li><li> 뇌가 명확하게 접근하면, 소뇌를 제거하고 PBS에 배치하는 집게를 사용합니다. 메스와 시상면에서 소뇌를 양분하고 4 ℃에서 하룻밤 30 % 자당 용액에 전송할 수 있습니다. 참고 : 수험하기파라핀 섹션입니다 전에 조직 매립 및 단면에 4 % PFA 4 ° C에서 하룻밤 소뇌를 해결. </li></ol> 4. 조직 가공 및 단면 <ol><li> 드라이 아이스, 2- 메틸 부탄의 슬러리를 조제. 욕조의 온도가 5 분 동안 평형을 허용합니다. </li><li> 10월 화합물과 cryomold를 입력하고 금형에 이분 소뇌의 절반을 배치합니다. </li><li> 해부 현미경과 집게 위치의 쌍 (소뇌의 중간 선 부근) 이분 된면이 위를 향하도록 소뇌 사용. </li><li> OCT의 화합물은 조직 표본 주위 백색 고체 매트릭스를 형성 할 때까지 5 분 동안 드라이 아이스 슬러리에 cryomold 옮긴다. 주의 : 필요하다면 냉동 시험편 지금 -20 ℃에서 저장 될 수있다. </li><li> 라벨 세르 15 μm의 단면을 수집하기 위해 제조자의 지시에 따라 조직 절편을 수집하기위한 폴리 리신 슬라이드를 처리 및 저온 장치가 미리 준비찾. 섹션을 수집하려면, 조심스럽게 슬라이드의 폴리 라이신 코팅 된면이 OCT의 섹션을 문의하십시오. 섹션의 방향을 유지하기 위해,이 단계에서 슬라이드를 회전하지 않습니다. </li><li> 염색 및 이미징을 진행하기까지 -20 ° C에서 보관 조직 섹션. </li></ol> 5. 염색과 소뇌 섹션의 이미지 <ol><li> 조직 염색하기 전에 다음과 같은 솔루션을 준비 : PBS의 ~ 50 ㎖ ~ PBS에서 4 % PFA의 5 ~ 10 ml의 ~ PBS에서 0.5 % 트리톤 X-100, 10 % 당나귀 혈청의 10 ~ 20 ml의 </li><li> 해동 소뇌 부분. 중요한 것은, EGFP의 퇴색을 방지하기 위해 지속적이고 강렬한 빛의 소스에 노출을 최소화 할 수 있습니다. </li><li> 소수성 장벽 펜을 사용하여 조직 섹션 주위에 사각형을 그립니다. </li><li> 5 분 동안 4 % PFA와 섹션을 포스트 - 수정. 소수성 장벽 (보통 ~ 500 μL)로 구분 전체 영역을 커버하기에 충분한 액체를 사용합니다. 진공 또는 피펫을 다시 사용정착은 조직 절편을 방해하지 않도록 특히주의하면서 이동합니다. </li><li> 피펫으로 부드럽게 조직 섹션을 방해하지 않고 슬라이드에 PBS를 적용합니다. 5 분 후 조심스럽게 액체를 제거하기 위해 진공 또는 피펫을 사용합니다. PBS가 3 회 반복하여 총 린스. </li><li> 피펫을 사용하여 조심스럽게 PBS에서 0.5 % 트리톤 X-100, 10 % 당나귀 혈청 (솔루션의 9​​00 μL 당 3 μL Calbindin 및 / 또는 Nesprin2 항체) 적절한 일차 항체의 희석을 적용합니다. 어둠 속에서 실온에서 1 시간 동안 배양한다. 주 : EGFP-KASH2은 동결 절편에 직접 형광 현미경으로 볼 수 있으며이 항 EGFP 항체를 이용할 필요가 없다. 파라핀 섹션이 준비되어있는 경우, EGFP-KASH2의 영상은 반 EGFP 항체와 면역을 필요로한다. </li><li> 항체 용액을 제거하고 PBS로 3 회 씻어. </li><li> 제조 1 : 0.5 % 트리톤 X-100-알렉사 형석 이차 항체 1,000 희석액,어둠 속에서 10 % 당나귀 PBS에서 혈청 및 저장. </li><li> 조심스럽게 최종 PBS 린스를 제거하고 어둠 속에서 한 시간 동안 차 항체 희석을 적용합니다. </li><li> 이차 항체 용액을 제거 씻어 절을 세 번 씻는다. 필요한 경우, 제 PBS의인가 등 DAPI (~ 300 ㎚) 등의 핵 염색을 적용한다. </li><li> 완전히 조직과 조직 섹션에 미디어를 장착 장소 ~ 5-7 μL 각국에서 PBS 용액을 제거합니다. 조직 절편 위에 유리 커버 슬립을 놓고 어둠 속에서 실온에서 ~ 15-30 분간 방치. </li><li> EGFP와 같이 5.8 / 5.9에서 사용 된 이차 항체에 적합한 임의의 다른 파장을 시각화하는 적절한 필터를 갖춘 형광 현미경을 사용한다. 소뇌 조각에서 EGFP-KASH2 핵 림을 보려면 20 배 목표를 사용합니다. </li></ol>

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저자가 공개하는 게 없다.

가장 중요한 단계는 성공적으로의 Tg (CAG-LacZ를 / EGFP-KASH2) 모델이 적합 Cre 호텔 마우스 라인 (들)을 식별하는 것입니다 사용하여 생체 내에서 LINC 복합체의 역할을 연구합니다. Cre 호텔 유사한 경​​로에 관여하는 다른 세포 유형에서 활성 상태 인 경우 사실, 그것은 결과의 해석을 복잡하게 할 수 있습니다. 소뇌 (도 2B)의 분자량 및 과립 세포층 여기에 도시 된 바와 같이, 따라?...

핵 봉투 (NE)는 세포질에서 핵질을 분리한다. 그것은 핵 구멍에 연결하는 내부 및 외부 핵 막 (INM 각각 ONM)로 구성되어있다. 모두 멤브레인로 구분 루멘은 핵 주변 공간 (PNS)라고합니다. ONM 러프 소포체 (ER)의 연장이고, INM 핵 박판에 부착, A- 및 B 형으로 표현 핵-V 형 중간 필라멘트 그물 세공은 1,2 라민. Nucleoskeleton 및 세포 뼈대 (LINC) 복합체의 링커는 물리적 골격 필라멘트 및 분자 모터 (그림 1A)에 핵의 내부를 연결하는 전체 핵 봉투에 걸쳐 거대 분자 어셈블리입니다. 일 (SAD1 / Unc84) 단백질과 Nesprins (핵 봉투 SPectRINS) : 그들은 NE의 통합 횡단 단백질의 두 가족의 특성을 진화 적으로 보존 모티브 사이의 상호 작용으로 구성되어 있습니다. 포유 동물에서, Sun1 Sun2에 ARN 말단 영역 nucleoplasmic A- 및 B 형 라민 3-5와 직접 상호 작용 INM의 E 막 횡단 단백질. PNS 내 INM의 다른 측면에서, 썬 단백질은 150 C- 말단 아미노산이 태양 도메인이라고 ~의 진화 - 보존 스트레칭 항구. SUN 도메인은 진화 - 보존 KASH와 직접 상호 작용 (Klarsicht / ANC-1, 영신의 인사 상동) 도메인, Nesprins의 분자 서명. KASH 도메인은 막 횡단 도메인 (6) 다음에 PNS 내로 돌출 ~ 30 C 말단 아미노산 스트레치로 구성된다. 적어도 네 가지 Nesprin 유전자 (Nesprin1-4)는 NE 7에서 지역화 단백질을 KASH 함유 인코딩. 놀라운 1,000 kDa의 (Nesprin1 거인)에 크기가 달라질 ~ 50kDa (Nesprin4)에서 Nesprins의 세포질 영역은 여러 스펙 트린은 액틴, plectin 및 분자 모터 8과 같은 세포 골격의 구성 요소와 상호 작용을 가능하게 반복뿐만 아니라 특정 주제 포함 (13). &lt;척추 동물과 무척추 동물의 P 클래스 = &quot;jove_content&quot;&gt; 연구 라민 / 일 / Nesprin / 분자 모터는 진화 적으로 보존 된 &quot;축&quot;핵 이동과 고정을 제어를 구성하는 것으로 나타났습니다. LINC 복잡한 구성 요소의 여러 녹아웃 (KO) 마우스 모델을 설명하고 포유류의 개발 9,14,15시 (NE)에서 태양과 Nesprin 단백질의 역할을 이해하는 프레임 워크를 제공하는 수단이되었다되고있다. 그러나, 이러한 모델 특히 현재 몇 가지 중요한 단점 :의 표현형 기여 구별 비 자치 효과, 2) 어려움을 세포에 의한 표현형 해석 1) 어려움 KASH없는 Nesprin 대 KASH이 함유, 3) 16 이소 폼 다양한 세포 유형의 NE에서 태양과 Nesprin 단백질의 기능 중복은 모두의 KASH 도메인에 대한 결핍 마우스 (17)의 모든 SUN-KASH 상호 작용과 마우스의 4) 주 산기 치사를 비활성화하는 복잡한 번식 체계를 필요Nesprins1 2는 성인의 분석 18 표현형을 배제한다. 이 프로토콜은 따라서 위에서 설명한 단점이 많은 우회, 셀 자율과 발달 규제 방식으로 생체 내에서 모든 Sun-KASH 상호 작용을 방해하기 위해 설계된 새로운 마우스 모델에 대해 설명합니다. 이는 Cre 호텔 / LOX 계 마우스 모델은 두 개의 중요한 개념에 의존한다 : 1) 공지 Nesprin 단백질 KASH 도메인은 세포 배양 시스템에서 NE에 EGFP 타겟팅 및 2) 일 도메인이 과발현 따라서 KASH 도메인으로 이것은 무작위 상호 작용하기에 충분한 어떤 KASH 도메인은 모든 내생 일 도메인을 포화과 지배적 인 음성 방식 (17) (그림 1B)에 LINC 복합체를 비활성화합니다. 이 프로토콜은 조직 수확 및 소뇌 조롱박 세포의 모든 SUN-KASH 상호 작용의 중단을 확인하는 데 사용 처리 단계를 설명합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Slides</td>
			<td>StatLab</td>
			<td>M1000W</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmuEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin Antibody</td>
			<td>Sigma Aldrich</td>
			<td>C9848</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EGFP Antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nesprin2 Antibody</td>
			<td></td>
			<td></td>
			<td>Previously described in Ref. 21</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>Sigma Aldrich</td>
			<td>O3551</td>
			<td></td>
		</tr>
	</tbody>
</table>

validation of a mouse model to disrupt linc complexes in a cell-specific manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

이 프로토콜의 Tg (PCP2-Cre 호텔) 생쥐를 사용 소뇌 조롱박 세포 EGFP-KASH2 발현을 제한 (TG-LacZ를 CAG / EGFP-KASH2) 마우스 모델의 유용성을 도시한다. Tg가에서 (PCP2Cre CAG-EGFP / KASH2) 자손, LacZ를 / V5 오픈 리딩 프레임함으로써 특히 조롱박 세포 (그림 2A)를 대상으로 EGFP-KASH2의 발현을 유도하는 Cre 호텔 재조합 효소에 의해 P6에서 절제되어있다. 예상대로 핵 (그림 2B)의 주위에...

Watch this Scientific Journal Video about Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner at JoVE.com

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

마우스 모델의 유효성 검사는 세포 고유의 방식으로 LINC 단지를 방해

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

validation-mouse-model-to-disrupt-linc-complexes-cell-specific

Research

JoVE Journal

Biology

7.3K 조회수.  Washington University in St. Louis School of Medicine. Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

비디오: Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

잠재적인 파킨슨병의 유전자 검증을위한 C. 엘레간스 도파민 신경 세포의 변성 분석의 응용

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

연구

생물학

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

CXC Chemokine 수용 체 4에 붙일 레이블 CXC Chemokine Ligand 12의 바인딩 중단 하는 화합물을 식별 하는 흐름 Cytometry 기반 시험

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

장 부분 방해의 마우스 모델

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

요추 척추 불안정의 마우스 모델

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

1차 배양에서 분화된 마우스 지방세포: 인슐린 저항성 모델

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...