우리 모두는 콜드 웰 연구소 회원의 협력 정신을 인정하고 싶습니다. 연구실에 운동 장애 연구는 바흐만 - 스트라우스 Dystonia &amp; 파킨슨병 재단, 미국 파킨슨병 재단, 미국 파킨슨 질병 협회, 앨라배마 파킨슨병 협회, 파킨슨병 연구에 대한 마이클 J. 폭스 재단과 대학 연​​구소에 의해 지원되었습니다

A. 식 플라스미드 건설 두 plasmids이 필요합니다 : 조직 특정 관심의 유전자의 표현과 선택 전환 표시로 두 번째 (플라스미드 마커가 연구 공동체에서 일반적으로 사용할 수 있습니다하지만) 한. 실험 플라스미드 <ol><li> 관심 조직 / 세포 유형에 표현되는 모터를 선택,이 경우, 검사의 수송은 (Pdat - 1) 발기인이 사용됩니다. 이 표현 플라스미드는 복제 recombinational 1 모터 관심 하류의 유전자의 삽입을 허용하는 Invitrogen 게이트웨이 시스템을 호환 대상 벡터 (pDEST - DAT - 1)로 만들어졌습니다. </li><li> 관심 유전자의 cDNA는 PCR이 게이트웨이 AT &amp; T B recombinational 시퀀스를 포함하는 primers를 사용하여 증폭됩니다. 증폭된 cDNA 먼저 입력 벡터를 만들 수있는 기증자 벡터 pDONR201에 recombined 후 E. 대장균 DH5α 변형으로 변환됩니다. 성공 recombinants과 DNA의 미니 준비 고립의 선택에 따라, 입력 벡터는 표현식이 플라스미드 만들 pDEST - DAT - 1 대상 벡터에 recombined입니다. recombinational 복제에 대한 자세한 내용은 Invitrogen 게이트웨이 설명서 중 또는 콜드웰 외에서 사용할 수 있습니다. 5. </li></ol> 플라스미드 선택 마커 플라스미드 형질 마커는 명확한 조직에서 형광 단백질의 표현을 구동 모터와 벡터로 구성되어 있습니다. 이되도록 특정 프로 시저에서, UNC - 54 발기인은 신체 벽 근육의 벚꽃 단백질 발현 (: 벚꽃 Punc - 54)을시킵니다. Microinjection 통해 트렌스 제닉 C. 엘레간스의 B. 생성 관련 조브 문서를 참조하십시오 <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833을</a> C. 유전자는 DA의 neurodegeneration 분석을위한 십자가 <ol><li> 웜 표준 절차 6을 사용하여 재배하고 있습니다. </li><li> 플레이스 10 Pdat - 1 : α - SYN, Pdat - 1 : 3-4 L4 단계 형질 전환 나온것과 짝짓기 접시에 GFP의 남성 1, 2 (: 유전자 X와 Punc - 54은 : Pdat - 1을 표현 : 체리) . 이것은 만들어진 세 개의 별도의 안정적인 라인 각각에 대해 개별적으로 수행되어야합니다. 이일 후 수컷을 제거합니다. </li><li> F1 세대를 검사, 여러 남성 자손이있다면, 교미를 성공적으로 완료했습니다. </li><li> 각 십자가에서 다섯 남녀 추니 L4 동물을 복제되는 전시 모두 Pdat - 1 : GFP 형광 마커 (남자 부모로부터 상속)과 Punc - 54 : 벚꽃 형광 바디 벽 근육 마커 (남녀 추니의 부모로부터 상속). 복제된 동물들은 아직 플레이트에있는 남자 동물 성관계를하지 않도록하기 위해 L4 단계에서해야합니다. 그들이 자기 ​​십자가와 F2 자손을 생산하도록 허용합니다. </li><li> 3 단계에서 생산 F2 동물 밖으로 복제됩니다. 특히, 전송 ~ 자신의 개인 접시에 두 형광 마커를 전시 5-10 동물. 화면 동물 표현 DA 뉴런에 GFP과 동물의 일부 100 % 신체 벽 근육 세포에 표현 벚꽃 아르 접시에 대한 F3 세대. 이러한 동물 Pdat - 1 homozygous 것입니다 : α - SYN, Pdat - 1 : GFP 유전자 안정 새로 만든 transgene (유전자 X)를 전송하는 동안. </li></ol> D. Dopaminergic 신경 세포 분석 <ol><li> 새로운 위에서 만든 세 줄의 각각의 접시뿐만 아니라 Pdat - 1을 준비 : α - SYN, Pdat - 1 : GFP 혼자 변형. 그들이 성인으로 성장할 수 있습니다. </li><li> 새로운 접시에 각 라인 30-40 형질 어른을 전송합니다. 그들은 20 º C. 4 시간 동안 태아를 낳기 위해 허용 </li><li> 어른을 제거합니다. 그들은 L4 단계에 도달할 때까지 배아가 20 º C에서 3-4일에 대한 개발을 허용합니다. </li><li> 0.04 MG / ML 5 - 플루오로 - 2&#39; - deoxyuridine (FUDR)이있는 접시로 ~ 100 유전자 변형 L4 동물을 선택하십시오. DNA 합성의 억제를 통해 다음 세대의 뉴클레오 티드 아날로그 블록 개발, 따라서 실험 동물 7 압도에서 자식을 방지. 그것이 가벼운 민감한이기 때문에 빛을에서 FUDR 번호판을 보호할 수 있습니다. </li><li> 성인 동물 계란 누워있는 다음과 같은 여러 일에 분석할 수 있습니다. 분석의 적절한 일은 경험적으로 결정됩니다. 예를 들어, 우리는 결정했다고 77%, 87 %, 그리고 Pdat - 1의 90 % : α - SYN, Pdat - 1 : GFP의 동물들이 각각, 일 6, 7 및 10 게시물 개발에서 퇴화한 검찰의 뉴런을 나타냅니다. 따라서, 우리가 관심의 transgene이 neurodegeneration을 강화 수도 예측한다면, 우리는 6 일 및 / 또는 7 분석합니다. 마찬가지로, neurodegeneration을 억제 수도 있습니다 transgenes은 7 일 10시 분석됩니다. </li><li> 4 신선한 아가로 오스 패드를 (microinjection의 한천 패드와는 달리, 이들은 신선한 사용하고 밖으로 건조 수 없습니다)합니다. 그들에 걸쳐 테이프의 조각을 두 가지 현미경 슬라이드를 설정하고, 테이프 equivalently 두꺼운 패드를위한 스페이서 역할을합니다. 3 분의 1을하다(그들은 세 나란히 위치에 있습니다) 벤치에서 그들 사이에 슬라이드. 중앙 슬라이드 용융 아가로 오스의 드롭을 장소와 빠르게 직각 세 슬라이드와 전체, 아가로 오스 상단에서 다른 슬라이드를하다. 패드를 사용할 준비가 될 때까지 함께 두 슬라이드를 떠나 여러 추가 패드하십시오. </li><li> 웜 검찰의 뉴런을 분석, 22 X 30mm 커버 유리 3 MM의 levamisole (마취)의 6 μl 방울을 넣으십시오. 아가로 오스 패드에 커버 유리 반전 후, 드롭으로 (10 30 이상의 추가 득점 수) 40 성인 동물을 선택하십시오. 다른 라인의 각각에 대해 반복합니다. </li><li> epifluorescence 및 FITC 또는 GFP의 시각화를 허용 필터 큐브와 복합 현미경을 사용하여 각 CEP 및 ADE 신경 세포의 존재에 대한 점수 줄에 30 동물. 우리는 GFP HYQ 필터 큐브를 (채도 기술) 부여를 사용하십시오. 첨부된 채점 시트에 기록 데이터입니다. 야생 동물 종류가 완료 모든 4 CEP에서 GFP의 형광 2 ADE의 뉴런을 유지합니다. DA의 뉴런의 손실을 전시 개별 동물은 아닌 WT 또는 &quot;degenerating&quot;로 득점 있습니다. 마찬가지로, 변성의 정도는 각 동물에서뿐만 아니라 인구로 잃은 총 뉴런를 세어 득점 수 있습니다. </li><li> 분석에게 두 번 이상 (시험 줄에 30 이상 동물)을 반복합니다. 분석의 3 라운드에서 야생 형 DA의 뉴런을 전시 웜의 총수는 평균입니다. neuroprotection에 대한 통계 분석은 다음 제어 웜 (: α - SYN, Pdat - 1 : Pdat - 1 : GFP)을 비교하는 학생의 T - 테스트 (P &lt;0.05)를 사용하여 수행됩니다 변종이있는 DA의 뉴런에 이상 - 특급 후보 유전자 . </li></ol>

<ol>
	<li>Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsin-mediated+neuroprotection+from+cellular+stresses+to+dopaminergic+neurons+of+C.+elegans.">Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans.</a> J Neurosci. 25, 3801-3812 (2005).</li><li>Hamamichi, S., Rivas, R. N., Knight, A. L., Cao, S., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis-based+RNAi+screening+identifies+neuroprotective+genes+in+a+Parkinson&#x2019;s+disease+model.">Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson&#x2019;s disease model.</a> PNAS. 105, 728-733 (2008).</li><li>Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-synuclein+blocks+ER-golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">alpha-synuclein blocks ER-golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J. -. C., McCaffery, J. M., Barlowe, C., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson's+disease+protein+alpha-synuclein+disrupts+cellular+Rab+homeostasis.">The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.</a> PNAS. 105, 145-150 (2008).</li><li>Caldwell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Genomics:+A+Discovery-Based+Laboratory+Course.">Integrated Genomics: A Discovery-Based Laboratory Course.</a> , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Gandhi, S., Santelli, J., Mitchell, J. D. H., Stiles, J. W., Sanadi, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+maintaining+large,+aging+populations+of+Caenorhabditis+elegans.">A simple method for maintaining large, aging populations of Caenorhabditis elegans.</a> Mechanisms of Ageing and Development. 12, 137-150 (1980).</li></ol>

도파민 뉴런의 나이에 의존 손실이 파킨슨병의 임상적 특징이며 알파 synuclein이라는 단백질의 축적이나 misfolding와 연결되었습니다. 여기 C.의 dopaminergic 신경, 형광등 transgene을 통해, 레이블하는 방법을 보여줍니다 엘레간스 이러한 세포의 coexpressing 인간 알파 synuclein에 의해 파킨슨병에서 본 neurodegeneration을 모방. 이 동영상은 성장, 유전자 교차점, 장착 및 중 neurodegeneration을 강화하거나 노화?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Agarose</td>
<td>Ultrapure</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>
<tr>
<td>Coverglass 20x30 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-548-5A</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Slides</td>
<td>Plain, 3x1"</td>
<td>Fisher</td>
<td>12-549</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting with Fluorescence</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ645</td>
<td>&nbsp;</td>
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<td>Epifluorescent</td>
<td>Microscope</td>
<td>Nikon</td>
<td>Model E-800</td>
<td>&nbsp;</td>
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<td>Filter Cube, GFP HYQ</td>
<td>Endow Bandpass</td>
<td>Chroma Technology</td>
<td>&nbsp;</td>
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application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes

파킨슨병 (PD)의 진단과 치료에 개선이 위험 인구를 렌더링 자화율 요인에 대한 지식에 의존하고 있습니다. 경찰과 관련된 새로운 유전자 요소를 식별하는 시도의 과정에서, 과학자들은 후보 유전자, polymorphisms, 중요한 진보를 대표하는 단백질의 많은 목록을 생성 있지만, 이러한 리드 mechanistically 정의되지 않은 상태로 유지됩니다. 우리 작품은 상당히 간단한 동물 모델 시스템의 장점을 이용하여 이러한 목록을 축소 방향으로 목표로하고 있습니다. 인간 뉴런 수십억을 가지고 있지만, 미세한 회 Caenorhabditis 엘레간스 정확하게 302을 가지고있는 hemaphrodites에서 단지 8 생산의 도파민 (DA). 투약 및 연령에 의존 neurodegeneration에 C. 엘레간스 DA 뉴런 결과에서 PD - 관련 단백질, 알파 synuclein를 인코딩 인간 유전자의 표현. DA의 뉴런 인간의 알파 synuclein을 표현하는 벌레는 isogenic하고 검찰의 전송 모터 (Pdat - 1)에서 GFP과 인간 알파 synuclein 모두를 표현한다. GFP의 존재이 동물에서 검찰의 neurodegeneration에 따라위한 쉽게 시각 마커 역할을합니다. 우리는 처음 알파 synuclein 유발 검찰 neurodegeneration가 torsinA, 분자 보호자 활동 1 단백질 이러한 동물 구출 수 있다고 보여주었다. 또한, 후보 PD 관련 유전자 그러면 C. 엘레간스 DA 뉴런의 과잉 표현했다 알파 synuclein의 misfolding 분석을 사용하여 대규모 RNAi 심사 노력을 통해 우리가 실험실에서 발견. 우리는 그들이 알파 synuclein - 유발 - DA neurodegeneration 2 구조로 다섯 일곱 유전자 PD의 대표 중요한 후보 변조기를 테스트 사실을 확인했습니다. 알파 synuclein에 의존 독성이 세포 독성을 강화하거나 억제 유전자에 대한 수 판독로 사용됩니다 의하여 또한, 린드 퀴 스트 연구소 (목성의 문제) 효모 화면을 수행했습니다. 우리는 이후의 C. 엘레간스 알파 synuclein - 유도 neurodegeneration 분석에 효모 후보 유전자 검사를하고 성공적으로 이러한 목표를 3, 4의 많은 검증. 우리의 방법론은 Pdat - 1 프로 모터의 제어하에 후보 유전자 cDNAs의 recombinational 복제를 사용하는 C. 엘레간스 DA 뉴런 특정 발현 벡터의 생성을 포함한다. 이러한 plasmids 그러면 성공적인 변환을위한 선택 마커와 함께 야생 타입 (N2) 벌레에 microinjected 있습니다. DA의 뉴런에서 후보 단백질을 생산하는 다수의 안정적인 유전자 변형 라인은 노화의 과정을 통해, 동물 및 개별 신경 세포 수준 모두에서, 취득 후 독립적으로 건너 알파 synuclein 퇴행성 변형에와 neurodegeneration에 대한 평가입니다.

Watch this Scientific Journal Video about Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes at JoVE.com

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

이 동영상은 파킨슨병에 대한 모델로 dopaminergic 신경 세포의 neurodegeneration을 평가 C. 엘레간스를 사용하는 방법을 보여줍니다. 또한, 유전자 화면은 어느 변성을 향상 또는 neuroprotective 가지 요인을 식별하는 데 사용됩니다.

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 ...

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

15.9K 조회수.  University of Alabama. 이 동영상은 파킨슨병에 대한 모델로 dopaminergic 신경 세포의 neurodegeneration을 평가 C. 엘레간스를 사용하는 방법을 보여줍니다. 또한, 유전자 화면은 어느 변성을 향상 또는 neuroprotective 가지 요인을 식별하는 데 사용됩니다.

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

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson: Development of New Therapies for Parkinson&#039;s Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

올 Isacson : 파킨슨병을위한 새로운 요법 개발

연구

생물학

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

파킨슨병에 대한 Murine 모델 : 6 - OH 도파민의 병변에서 행동 테스트

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

트렌스 제닉의 생성 C. 엘레간스 Biolistic 변환에 의해

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

생산 C. 엘레간스</em함께 Recombineering 통해> Transgenes galK 선택 마커

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics : 수치과 구조 변경의 탐지는 모두 24 인간의 염색체로 동시에 소설 OctoChrome 생선 분석을 사용

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

선충류의 바디 크기 규정에 참여 유전자에 대한 화면으로 RNA로 인한 간섭 수유 전략을 사용하여<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans의 주 화성 분석

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

배아 배양하는 방법<em&gt; C. 엘레</em&gt; 셀

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

에 박테리아의 효과를 측정<em&gt; C. 엘레</em계란 보존 분석을 사용하여&gt; 행동

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...