The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.

LED가 조명기 1. 건설 주 : 필요한 장비는 LED 자재 목록에 요약 하였다. 전체 LED 설치가 작고, 우리는이 웜 (4)의 빛 노출을 제어하는 어두운 방에 배치 할 것을 권장하지만, 실험실에서 삽입 될 수 있습니다. <ol><li> 케이블 (그림 1A, D)와 컨트롤러에 LED를 연결합니다. </li><li> BNC 케이블 (그림 1A, C, D)와 디지털 함수 발생기 / 앰프 컨트롤러를 연결합니다. </li><li> 사용자 정의 홀더 (그림 1A)를 사용하여 스테이지 위의 LED 조명 10cm를 수정합니다. 참고 : 우리는 금속 부품 홀더를했다. </li><li> 부분적으로 LED 조명 설치를 커버하지만, 웜 아프게 축열 (그림 1B)를 피하십시오. 참고 : 우리는 개방 상단과 하단 (그림 1A)와 주문 제작 하드 플라스틱 커버를 사용합니다. 커버는 돌연변이 자체에 요구되지 않으며, t를 사용오 주위에 푸른 빛의 불필요한 노출을 제한합니다. 우리는 벌레의 과열을 방지하기 위해 LED 설치 완전히 (그림 1B)를 포함하지 않는 것이 좋습니다. </li><li> 레이저 보호 안경을 착용 할 것. 주 : 빛이 매우 밝은이기 때문에 우리는 레이저 보호 안경을 착용하십시오. 선글라스가 잘 작동 할 수 있습니다. </li><li> 에 LED 컨트롤러의 레버 스위치 &#39;지능을.&#39; 연속 조명 (그림 1D)의 최대 전력 (그림 1C)의 65 %로 설정합니다. </li><li> 벌레는 제조 업체의 프로토콜을 사용하여 배치 단계에있는 푸른 빛의 강도를 측정하는 광도계를 사용합니다. 설정은 연속 조명 아래 2.0 mW의 / mm 2를 제공합니다. </li></ol> 2. 블루 라이트 치료 참고 :. 인증 기관은-UNC가-119 (+) Pmex-5-HIS-72 : miniSOG - 3&#39;UTR (TBB-2) +] UNC-119 (ED3) III 4 균주를 사용할 수있는 변형 CZ20310 juSi164를 사용하여 Caenorhabditis 엘레 유전학 센터 (CGC에서, HTTP: //www.cgc.cbs.umn.edu/). <ol><li> E. 표준 선충의 성장 배지 (NGM)에 어둠 속에서 그의-mSOG의 피로를 유지 판 대장균 OP50 표준 프로토콜 10, 11 다음. 참고 : 주변 광에 따라, juSi164 함유 균주가 자발적 비율 4 위의 돌연변이 속도를 표시하지 않습니다. 일반적뿐만 아니라 돌연변이를 일으키지 않는 할로겐 램프와 해부 범위를 사용 루틴 변형 통로. 그럼에도 불구하고, 치료는 표준 어두운 내부 배양기에서 긴장을 유지, 또는 호일로 변종을 포함, 예를 들어 빛에 불필요한 노출을 피하기 위해주의해야한다. 불필요한 돌연변이가 시간이 지남에 축적 경우를받은 후 균주의 분취 량을 동결하는 것이 좋습니다. </li><li> 임신하는 젊은 성인을 선택 웜으로 (약 12 시간 후 L4) 동물 (11)을 선택합니다. 참고 : 오래된 동물이 낮은 무리의 크기를 가질 수있는 동안 젊은 동물, 이하 돌연변이 유발 효과 4을 보여줍니다. </li><li> 필터 페이지를 잘라조리개는 25mm X 25mm 사각형 구멍이있는 60mm 판에 맞게 및 시드 화 판 (그림 1E)에 배치합니다. 주 : 사각형 펀치를 사용할 수 있지만, 펀치의 크기가 정확하지 않습니다. </li><li> 접시에 사각형 구멍에서 벌레를 제한 균등 필터 종이에 100 밀리미터의 CuCl 2의 100 μL를 삭제합니다. </li><li> 임신하는 젊은 성인 자웅 동체의 원하는 번호를 (예를 들어 3 단계 참조) 선택과의 CuCl 2 판의 중심에 전송할 수 있습니다. </li><li> 레이저 보호 안경을 착용 할 것. </li><li> LED와 함수 발생기 ON 전환합니다. </li><li> 에 LED 컨트롤러의 레버 스위치 &#39;내선을.&#39; 함수 발생기 (도 1D)가 그것을 제어한다. </li><li> 최대 전력의 65 %에서 컨트롤러와 사인파, 4 Hz에서 (그림 1C)와 같은 함수 발생기를 설정합니다. </li><li> LED가 (그림 1A &lt;아래 단계에 뚜껑없이 단계 2.5에서의 CuCl 2 판을 배치/ STRONG&gt;). </li><li> 30 분 동안 푸른 빛으로 동물을 조명. </li><li> P 0으로 시드 판에 건강한 찾고 웜을 전송합니다. 참고 : P 0의 원하는 번호가 화면의 종류에 따라 다릅니다. 예를 들어 3 단계를 참조하십시오. 는 P 0 벌레는 빛 치료 후 즉시 조정되지 않은 것으로 나타날 수 있으며, 시간이 지남에 따라 회복됩니다. </li><li> 인큐베이터이나 어둠 속에서 호일을 사용하여 적용 벌레를 유지; 그들의 자손 2,10 중 원하는 표현형을 심사 시작하는 표준 절차를 따르십시오. </li></ol> 순방향 유전 화면 3. 예 참고 :. 이것은 LED 및 변형이 작동하는지 확인하기 위해 120 반수체 게놈을 가진 클론 화면의 예입니다 그림 2는 절차의 개략도를 보여줍니다. <ol><li> [첫째날] 푸른 빛을 처리 한 후, P 0으로 OP50와 NGM 판에 다섯 건강한 찾고 벌레를 선택 20 ° C 배양기에서 그들을 유지하고 그들에게 1 알을 낳기하자D ( &#39;첫째날&#39;판). </li><li> [둘째 날] 새 시드 판에 전송 P 0 ( &#39;둘째 날&#39;판). </li><li> [Day3] 선택하고 레코딩하여 &#39;둘째 날&#39;접시에 P 공을 죽일. </li><li> [Day4]를 &#39;첫째날&#39;판에서 30 임신하는 젊은 성인 F 1을 선택하고 개별 플레이트 ( &#39;첫째날&#39;30 F 1 판)에 전송할. </li><li> &#39;둘째 날&#39;플레이트 [Day5] 단계를 반복 3.4 ( &#39;둘째 날&#39;30 F 1 판). </li><li> 그들이 성인이 될 때 Day7-9] 표준 실체 현미경 하에서 비정상적인 형태 학적 및 / 또는 WT 변형 (그림 3A)에 비해 F 2 웜의 동작을 확인합니다. 참고 : 높은 침투와 열성이다 F 2 중 같은 표현형과 벌레의 내무반에 관한 유전 변이가 표시됩니다. 그러나 일부 돌연변이 천천히 성장 및 / 또는 부분적인 침투를 표시 할 수 있습니다. 따라서, 돌연변이 미만 분기가 발견 될 수있다플레이트. </li><li> 개별적으로 볼 수 표현형과 벌레를 선택하고 다음 세대의 표현형의 유전을 확인합니다. </li></ol> 염색체 외 형질 전환 유전자 통합 4. 예 <ol><li> A의 CZ20310 juSi164에 관심있는 DNA를 주입 [Pmex-5-HIS-72 : miniSOG - 3&#39;UTR (TBB-2) + CB-UNC-119 (+)] UNC-119 (ED3) 및 준비 유전자 변형 벌레 표준 주입 프로토콜 12,13 다음 저 전송률 (10~30%). 참고 : 또는 설립 염색체 외 배열이 juSi164 균주에 교차 할 수 있습니다. juSi164에 대한 유전자형을 방지하기 위해, juSi164의 이형 효율이 감소 될 수 있지만 P 0으로 사용될 수있다. juSi164 대한 유전자형 방법은 섹션 5에서 설명한다. </li><li> 2 단계에 설명 된대로 [첫째날] ~ 푸른 빛 P 0로 (20) 형질 전환 동물을 취급합니다. 참고 : 더 P 0 웜은 그럴 필요에 따라 필요<html> ansmission 속도. 표현형은 유전자 또는 공사 출 마커 (12, 13)에 의해 식별 될 수있다. </li><li> 개인 접시에 10 건강한 찾고 P 0 선택 20 ° C 배양기에서 그들을 유지하고 며칠 (P 0 판)에 대한 알을 낳는하자 </li><li> [Day4] 싱글 아웃 (20) 형질 전환 F 1 개별 플레이트 상에 각각 P 0 판에서 벌레와 그들이 알을 낳기 수 있습니다. (20 F 1 × P 0 판 = 총 200 F 1 판). </li><li> [Day7] F에게&gt; 75 % F 2 가지는 유전자 1 판을 찾습니다. </li><li> F에게&gt; 75 % 전송 속도에서 1 판 (F 2 판) 오 형질 전환 동물을 선택합니다. </li><li> [Day10] F 잠재적 인 통합 선으로 100 %의 전송 속도 2 판을 보관하십시오. </li><li> 멘델의 분리를 확인하고 잠재적 인 배경 돌연변이를 제거하기 위해 표준 절차를 사용하여 (10) 전위 집적 라인 교배. </li></ol>제작 &quot;&gt; 5. 유전자형 <ol><li> 를 Lyse (20)는 표준 프로토콜 (14)을 사용하여 용해 완충액 F 2 웜 outcrossed. </li><li> 표준 프로토콜 14 다음 58 ℃의 어닐링 온도 MosSCI 삽입 9 juSi164 (표 1)에 대한 프라이머를 사용하여 PCR을 수행한다. 주 : UNC-119 (ED3)가 다음 단계 5.4 및 5.5에서 구하고 트랜스 (juSi164)을 제거한 후 배위 동작에 의해 검출 될 수 있기 때문에이 UNC-119 (ED3)를 유전자형 불필요하다. </li><li> 아가로 오스 겔 전기 영동 (15)을 실행하고 juSi164에 대한 WT를 찾기 위해 밴드 크기 (표 1)를 검사합니다. </li><li> 은 F 2 자손으로 인해 UNC-119 (ED3)의 존재에 벌레를 마비 경우 검사합니다. </li><li> 조정되지 않은 웜 단계 5.4에서 발견하는 경우, 하나의 여러 nonuncoordinated 웜은 다음 세대의 모든 nonuncoordinated 웜을 얻었다. </li></ol>

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J., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CloudMap:+A+Cloud-Based+Pipeline+for+Analysis+of+Mutant+Genome+Sequences.">CloudMap: A Cloud-Based Pipeline for Analysis of Mutant Genome Sequences.</a> Genetics. 192 (>4), 1249-1269 (2012).</li><li>Qi, Y., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiniSOG-mediated+Photoablation+in+Caenorhabdtis+elegans.">MiniSOG-mediated Photoablation in Caenorhabdtis elegans.</a> Bio-protocol. 3 (1), 316 (2013).</li><li>Qi, Y. B., Garren, E. J., Shu, X., Tsien, R. Y., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-inducible+cell+ablation+in+Caenorhabditis+elegans+using+the+genetically+encoded+singlet+oxygen+generating+protein+miniSOG.">Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG.</a> Proc Natl Acad Sci U S A. 109 (19), 7499-7504 (2012).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279-2284 (2005).</li></ol>

The authors have nothing to disclose.

여기서는 optogenetic 돌연변이의 상세한 절차는 그의-mSOG는 배아에서 발현 이용하여 소규모 순방향 유전 화면의 일례를 설명 제공. 표준 화학적 돌연변이 유발에 비해,이 optogenetic 돌연변이 유발 방법은 여러 가지 장점을 갖는다. 첫째, 이에 거리와 같은 EMS, ENU 같은 독성 화학 돌연변이에서 실험실 인력을 유지하고 자외선 (TMP / UV)와 trimethylpsoralen, 무독성이다. 둘째, 돌연변이 유발 실험 절차는 P ...

앞으로 유전 화면이 널리 예쁜 꼬마 선충 (C. elegans의) 1과 모델 생물의 다양한 생물학적 과정을 방해 유전 적 돌연변이를 생성하는 데 사용되었다. 이러한 돌연변이의 분석은 기능적으로 중요한 유전자의 발견을 유도 및 시그널링 1,2 경로. 전통적으로, C.에서 돌연변이 유발 엘레은 돌연변이 유발 화학 물질, 방사선, 또는 트랜스포존 2를 사용하여 달성된다. 에틸 메탄 (EMS) 및 N - 에틸 N의 -nitrosourea (ENU) 인간에게 독성이 같은 화학; 감마선 또는 자외선 (UV) 방사선 돌연변이 특별한 장비를 필요로; 및 뮤 테이터 균주 3 등 트랜스포존 활성 균주, 유지 보수시 불필요한 돌연변이를 일으킬 수 있습니다. 우리는 유 전적으로 인코딩 된 감광제 (4)를 이용하여 유전 변이를 유도하는 간단한 방법을 개발했습니다. 반응성 산소 종 (ROS)의 DNA 5가 손상 될 수 있습니다.미니 중항 산소 발생기 (miniSOG)는 LOV (빛, 산소 및 전압)에서 설계 한 106 아미노산 애기 phototropin 2/6 도메인의 녹색 형광 단백질이다. 청색광 (~ 450 ㎚), miniSOG에 노출시 ROS는 모든 세포에 존재하는 보조 인자 6-8로 FL AVIN 모노 뉴클레오티드와 일 중항 산소를 포함한 생성한다. 우리는 히스톤-(72), C의 C 말단에 miniSOG에 태그를 지정하여 그의-mSOG 융합 단백질을 구성 히스톤 (3)의 엘레 변형 우리는 C.의 생식 세포에 그의-mSOG을 표현하는 단일 복사 유전자 (9)을 생성 엘레. 어둠 속에서 정상 배양 조건은 그의-mSOG 유전자 변형 웜은 정상 무리의 크기와 수명 4 있습니다. 블루 LED 빛에 노출되면, 그의-mSOG 웜은 자신의 자손 4 사이의 유전 변이를 생산하고 있습니다. T에 C : A와 G : C에 C : G, 작은 chromoso에 의한 돌연변이의 스펙트럼은 G와 같은 염기 변화를 포함날 4 삭제. 이 돌연변이 유발 절차를 수행하는 간단하고, 최소한의 실험 안전 설정을 요구한다. 여기, 우리는 optogenetic 돌연변이 유발을위한 LED 조명 설정 및 절차에 대해 설명합니다.

<table><tbody><tr><td>Ultra High Power LED light source</td><td>Prizmatix</td><td>UHP-mic-LED-460</td><td>460 &amp;plusmn; 5 nm</td></tr><tr><td>LED controller</td><td>Prizmatix</td><td>UHPLCC-01</td><td></td></tr><tr><td>Digital function generator/amplifier</td><td>PASCO</td><td>PI-9587C</td><td>PI-9587C is no longer available. The replacement is PI-8127.</td></tr><tr><td>BNC cable male/male</td><td>THORLABS</td><td>CA3136</td><td></td></tr><tr><td>USB-TTL interface</td><td>Prizmatix</td><td></td><td>Optional</td></tr><tr><td>Photometer</td><td>THORLABS</td><td>PM50 and Model D10MM</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1001-110</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>NGM plate</td><td></td><td></td><td>Dissolve 5 g NaCl, 2.5 g Peptone, 20 g Agar, 10 &amp;micro;g/mL cholesterol in 1 L H&lt;sub&gt;2&lt;/sub&gt;O. After autoclaving, add 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 25 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;(pH 6.0).&amp;nbsp;</td></tr><tr><td>Lysis solution</td><td></td><td></td><td>10 mM Tris(pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 100 &amp;micro;g/mL Proteinase K</td></tr></tbody></table>

optogenetic random mutagenesis using histone-minisog in c. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

우리는 120 돌연변이 반수체 게놈에 해당하는 60 F 1 판 중 섹션 2와 3에 설명 된 표준 프로토콜 다음 30 분 빛 노출을 사용 CZ20310 juSi164 UNC-119 (ED3)와 앞으로 유전자 화면을 수행, 우리는 8 F 1 판을 발견 F 등의 신체 사이즈 결함 (그림 3B), 조정되지 않은 운동 (그림 3C), 성인 치사 (그림 3D)으로 볼 수 표현형을 표시?...

Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

유전자 인코딩 히스톤 - miniSOG는 푸른 빛 의존적으로 게놈 전체의 유전 변이를 유도한다. 이 돌연변이 유발 방법은 빠르고 간단 독성 화학 물질의 자유, 앞으로 유전자 검사 및 유전자 통합에 적합합니다.

히스톤 - miniSOG에서 사용 Optogenetic 무작위 돌연변이 유발<em&gt; C. 엘레</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Classical and Modern Genetics

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9.0K 조회수.  University of California in San Diego and Howard Hughes Medical Institute.  유전자 인코딩 히스톤 - miniSOG는 푸른 빛 의존적으로 게놈 전체의 유전 변이를 유도한다. 이 돌연변이 유발 방법은 빠르고 간단 독성 화학 물질의 자유, 앞으로 유전자 검사 및 유전자 통합에 적합합니다. 

비디오: Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

대상<em&gt; 현장에서</em신진 효모에서&gt; 돌연변이 유발 히스톤의 유전자

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

연구

유전학

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

분열 효 모 섬으로 불안정 프로테아좀 돌연변이 선택할 임의의 Mutagenesis 유전자 타겟팅

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

빠른 고 손쉬운 파이프라인 C. 선 충 을 사용 하 여 CRISPR/Cas9 Ribonucleoproteins에서 게놈 포인트 돌연변이 생성에 대 한

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

C. 선 충 에서 신경 세포를 세균 세포를 재 설 정할 RNAi 및 열 충격 유도 하는 전사 인자 식의 적용

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

발생학

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.의 생체 Neuronal 칼슘 이미징에서 elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

신경과학

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

웜 실험실 소개 : Culturing 웜에서 돌연변이 유발하는

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

생물학

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

이미징 C. 엘레간스</emEpifluorescent 현미경과 오픈 소스 소프트웨어를 사용> 태아

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

꼬마 선 충 나이 연구 변화 내재 단백질 집계에 방법

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans

Source: Groh, N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56464/methods-to-study-changes-inherent-protein-aggregation-with-age">Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp.&#160;(2017).
RNAi is powerful genetic tool available to C. elegans researchers. This video introduces a method to treat more worms with RNAi than plate feeding by utilizing liquid culture conditions.

액체 배양에서 RNAi 공급: 예쁜꼬마선충에서 유전자 발현을 knockdown하는 고처리량 방법

Source: Groh, N. et al. Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2017).
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히스톤 - miniSOG에서 사용 Optogenetic 무작위 돌연변이 유발

요약

더 많은 비디오 탐색

이 비디오의 챕터

대상

분열 효 모 섬으로 불안정 프로테아좀 돌연변이 선택할 임의의 Mutagenesis 유전자 타겟팅

빠른 고 손쉬운 파이프라인 C. 선 충 을 사용 하 여 CRISPR/Cas9 Ribonucleoproteins에서 게놈 포인트 돌연변이 생성에 대 한

C. 선 충 에서 신경 세포를 세균 세포를 재 설 정할 RNAi 및 열 충격 유도 하는 전사 인자 식의 적용

C.의 생체 Neuronal 칼슘 이미징에서 elegans

웜 실험실 소개 : Culturing 웜에서 돌연변이 유발하는

이미징 C. 엘레간스 태아

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

꼬마 선 충 나이 연구 변화 내재 단백질 집계에 방법

액체 배양에서 RNAi 공급: 예쁜꼬마선충에서 유전자 발현을 knockdown하는 고처리량 방법