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>

<ol>
	<li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+and+design+of+genetic+screens:+caenorhabditis+elegans.">The art and design of genetic screens: caenorhabditis elegans.</a> Nat Rev Genet. 3 (5), 356-369 (2002).</li><li>Kutscher, L. M., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+mutagenesis+in+C.+elegans.">Forward and reverse mutagenesis in C. elegans.</a> WormBook. , 1-26 (2014).</li><li>Collins, J., Saari, B., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+a+transposable+element+in+the+germ+line+but+not+the+soma+of+Caenorhabditis+elegans.">Activation of a transposable element in the germ line but not the soma of Caenorhabditis elegans.</a> Nature. 328 (6132), 726-728 (1987).</li><li>Noma, K., 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=Optogenetic+mutagenesis+in+Caenorhabditis+elegans.">Optogenetic mutagenesis in Caenorhabditis elegans.</a> Nat Commun. 6, 8868 (2015).</li><li>Cooke, M. S., Evans, M. D., Dizdaroglu, M., Lunec, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+DNA+damage:+mechanisms,+mutation,+and+disease.">Oxidative DNA damage: mechanisms, mutation, and disease.</a> FASEB J. 17 (10), 1195-1214 (2003).</li><li>Shu, X., 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=A+genetically+encoded+tag+for+correlated+light+and+electron+microscopy+of+intact+cells,+tissues,+and+organisms.">A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.</a> PLoS Biol. 9 (4), 1001041 (2011).</li><li>Ruiz-Gonzalez, R., 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=Singlet+oxygen+generation+by+the+genetically+encoded+tag+miniSOG.">Singlet oxygen generation by the genetically encoded tag miniSOG.</a> J Am Chem Soc. 135 (26), 9564-9567 (2013).</li><li>Pimenta, F. M., Jensen, R. L., Breitenbach, T., Etzerodt, M., Ogilby, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-dependent+photochemistry+and+photophysics+of+"miniSOG,"+a+protein-encased+flavin.">Oxygen-dependent photochemistry and photophysics of "miniSOG," a protein-encased flavin.</a> Photochem Photobiol. 89 (5), 1116-1126 (2013).</li><li>Frokjaer-Jensen, C., 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=Random+and+targeted+transgene+insertion+in+Caenorhabditis+elegans+using+a+modified+Mos1+transposon.">Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.</a> Nat Methods. 11 (5), 529-534 (2014).</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 (1), 71-94 (1974).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. (47), (2011).</li><li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+transgenic+C.+elegans+using+microinjection.">Generation of stable transgenic C. elegans using microinjection.</a> J Vis Exp. (18), (2008).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Fay, D., Bender, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mapping+and+manipulation:+chapter+4--SNPs:+introduction+and+two-point+mapping.">Genetic mapping and manipulation: chapter 4--SNPs: introduction and two-point mapping.</a> WormBook. , 1-7 (2006).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li><li>Westberg, M., Holmegaard, L., Pimenta, F. M., Etzerodt, M., Ogilby, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+an+efficient,+genetically+encodable,+protein-encased+singlet+oxygen+photosensitizer.">Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer.</a> J Am Chem Soc. 137 (4), 1632-1642 (2015).</li><li>Xu, S., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+optogenetic+cell+ablation+in+C.+elegans+using+membrane-targeted+miniSOG.">Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG.</a> Sci Rep. 6, 21271 (2016).</li><li>Mariol, M. C., Walter, L., Bellemin, S., Gieseler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+protocol+for+integrating+extrachromosomal+arrays+with+high+transmission+rate+into+the+C.+elegans+genome.">A rapid protocol for integrating extrachromosomal arrays with high transmission rate into the C. elegans genome.</a> J Vis Exp. (82), (2013).</li><li>Lin, J. Y., 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=Optogenetic+inhibition+of+synaptic+release+with+chromophore-assisted+light+inactivation+(CALI).">Optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (CALI).</a> Neuron. 79 (2), 241-253 (2013).</li><li>Minevich, G., Park, D. S., Blankenberg, D., Poole, R. 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.

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			<td height="42" style="height:42px;width:256px;">Ultra High Power LED light source</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHP-mic-LED-460</td>
			<td style="width:184px;">460 &#177; 5 nm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LED controller</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHPLCC-01</td>
			<td style="width:184px;"></td>
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			<td height="63" style="height:63px;width:256px;">Digital function generator/amplifier</td>
			<td style="width:71px;">PASCO</td>
			<td style="width:115px;">PI-9587C</td>
			<td style="width:184px;">PI-9587C is no longer available. The replacement is PI-8127.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">BNC cable male/male</td>
			<td style="width:71px;">THORLABS</td>
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			<td height="21" style="height:21px;width:256px;">USB-TTL interface</td>
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			<td style="width:184px;">Optional</td>
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			<td height="42" style="height:42px;width:256px;">Photometer</td>
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			<td height="21" style="height:21px;width:256px;">Filter paper</td>
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			<td style="width:115px;">1001-110</td>
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			<td height="46" style="height:46px;width:256px;">Copper chloride dihydrate (CuCl2&#8729;2H2O)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:115px;">C6641</td>
			<td style="width:184px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Stereomicroscope</td>
			<td style="width:71px;">Leica</td>
			<td style="width:115px;">MZ95</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="163">
			<td height="163" style="height:163px;width:256px;">NGM plate</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 &#181;g/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0).&#160;</td>
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		<tr height="88">
			<td height="88" style="height:88px;width:256px;">Lysis solution</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 &#181;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 ...

optogenetic-random-mutagenesis-using-histone-minisog-in-c-elegans

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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

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

의 Transcriptomic 분석<em&gt; C</em&gt;.<em&gt; 간스</em갤럭시 프로젝트에 턱시도 제품군을 통해&gt; RNA 시퀀싱 데이터

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Chromatin Immunoprecipitation을 이용한 c-KIT 리간드 프로모터 분석

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

Cpf1을 이용한 C-Brick DNA 표준 어셈블리 프로토콜

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

C. 선 충 에서 광범위 한 범위 식이 제한 유전자 식 수준 동안 수명 변조에서 인코딩된 정보의 정량화

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

꼬마 선 충 에 Ploidy의 조작

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

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 ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Bemisia tabaci Cytogenetic 기술을 사용 하 여 계란 수정 속도 결정

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

단일 및 벌크 C.의 고처리량 정량적 RT-PCR. 나노 유체 기술을 사용하여 샘플

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

히스톤 - miniSOG에서 사용 Optogenetic 무작위 돌연변이 유발

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