우리는 원고에 도움이 코멘트 대표 결과 그림 3과 제이슨 와츠와 크리스 웹스터에 표시된 예비 실험을 수행하기 위해 크리스 웹스터 감사합니다. 이 연구를위한 자금 조달은 JLW에 건강의 국립 연구소 (미국) (R01DK074114)에서 교부금에 의해 제공되었다. 이 작품에 사용 된 일부 선충 균주 연구 인프라 프로그램의 NIH 사무소 (P40의 OD010440)에 의해 재정 지원 Caenorhabditis의 유전학 센터에 의해 제공되었다.

고도 불포화 지방산은 열, 빛과 산소에 민감합니다. 지방산의 보충 판을 준비 할 때 따라서주의가 필요 지방산 과잉 열과 빛에 노출되지 않도록. 0.1 % TERGITOL (NP-40)를 함유 NGM 매체 지방산 나트륨 염은 일정한 교반하면서 첨가 된 후, 오토 클레이브와 부분적으로 냉각된다. 플레이트는 어둠 속에서 건조 할 수있다. C.에 의해 지방산의 흡수 다음, 가스 크로마토 그래피로 모니터링 할 수있다 elegans의 이러한 플레이트 상에 배양 하였다. 1. 지방산 보충 미디어의 제조 <ol><li> 적절한 크기의 플라스크에 미디어 구성 요소를 측정한다. 1 L 당 17g 박토 한천, 2.5 g의 트립 톤, 3g의 NaCl, 에탄올 1 ㎖ 5 ㎎ / ㎖의 콜레스테롤, 물에 녹여 10 ㎖의 10 % TERGITOL를 추가합니다. </li><li> 밀리 포아 물 최종 원하는 볼륨의 80 %를 추가하고 다른 수와 동일 (빈 유리 병들과 함께 미디어를 압력솥지방산 피 시험 농도)뿐만 아니라, 적절한는 눈금 실린더. </li><li> 55 ° C로 설정 물을 욕조에 차가운 미디어 매체는 유리 바이알을 오픈 깨는 안전 예방책을 사용하고 유리 입자가 지방산과의 혼합하지 않는 보장함으로써 지방산 나트륨 염 작업 원액을 제조 냉각된다. 100 ㎜ 작업 주식을 할 수있을 정도로 충분한 지방산을 달다. </li><li> 정제수 100 ㎜의 최종 농도 지방산 해결책을 가져올. 완전 지방산 나트륨 염을 용해하는 것은 전형적으로 약 20-30 분 걸린다. 불활성 가스와의 접촉은 산화를 방지하기 때문에, 아르곤 또는 질소와 지방산의 작업 스톡 퍼지. 유리 병 캡 미디어가 식을 때까지 어둠 속에서 작업 주식을 저장합니다. </li><li> (선택 사항) RNAi의 매체가 요구되는 경우, 암피실린을 측정하고 여기에 이소 프로필 β-D-1-티오 갈 락토 피 라노 사이드 (IPTG) 해법. </li><li> 한천은 55 ℃로 냉각되면 1 L 당 추가 C : 11 M 망초의 ML, 1 M 염화칼슘 1 ㎖ 및 인산염 완충액 25 ㎖ (1 L 당 108.3 g의 KH 2 PO 4와 35.6 g의 K 2 HPO 4 멸균). 필터 살균 암피실린 및 IPTG 솔루션 RNAi의 판을 만드는 경우를 추가합니다. 플레이트의 모든 유형의 경우, 1 L.로 최종 미디어 볼륨을 가지고 멸균 물을 </li><li> 불 가까이, 멸균에 미디어를 전송하고 적절하게 눈금 실린더 크기의 다음 원하는 볼륨에 따뜻한 멸균 물을 추가합니다. 다시 초기 플라스크에 미디어를 전송하고 교반 혼합한다. </li><li> 화염 가까이에, 오토 클레이브로 측정하여 테스트 할 농도의 수와 같은 병의 번호로 미디어를 전송하고 적절하게 눈금 실린더 크기. 지방산 작업 재고가 완전히 용해 될 때까지 수욕을 사용하여 액체로 분주 미디어를 유지한다. </li><li> 미디어 터치 따뜻한,하지만 섹시하지가 될 때까지 저어 접시와 혼란에 하나의 병을 놓습니다. 충분히 자신을두고 있는지 확인지방산 원액에 저어 미디어가 응고를 시작하기 전에 플레이트를 넣는 시간. 볶음 판은 온도 제어가있는 경우 55 ° C.로 설정 </li><li> 원하는 최종 농도의 용지에 100 ㎜ 지방산 작업 주식을 희석. 볶음 미디어는 흰색 침전물 때까지 용액 (약 1 분)에 있습니다. 미디어는 여전히 그 후 약간 흐린 수 있습니다. </li><li> (선택 사항) RNAi의 미디어가 2 mM의 최종 농도 ㎖ 및 IPTG 0.1 ㎎ /에 추가 암피실린을 원하는 경우. </li><li> 8 60mm 판 당 미디어의 ML 100 mm 플레이트 당 미디어 25 ㎖를 추가, 자동 피펫 원조와 멸균 25 ML 피펫을 사용하여 미디어를 붓는다. 나머지 병의 단계를 쏟아 지방산 또한 플레이트를 반복합니다. </li><li> 한천은 고형화 후, 광 산화로부터 보호하기 위해 잘 배출 카세트 실온 상자 또는 저장소와 지방산 보충 플레이트를 커버. </li><li> 씨 E. 대장균 OP50 접시 건조 한 지 이틀 판에.60mm 판의 경우, (37 ° C에서 성장) 하룻밤 OP50 문화의 피펫 300 μL. RNAi의 플레이트가 부어 된 경우 플레이트가 나흘 동안 건조 후, RNAi의 박테리아의 300 μL와 씨앗. 플레이트 건조 시간으로 인해 다양한 실험실 환경에서 습도의 차이에 따라 조정해야 할 수도 있습니다. </li><li> 세균 잔디가 건조되는 동안 실온에서 어두운 환경에서 번호판을 품어. </li></ol> 2. DGLA의 보충에 의해 생식 세포의 파괴를 유도 <ol><li> C. 0.3 mM의 DGLA (20시 3분 N-6)을 포함 접시에 성장 엘레 인해 모두 애벌레 단계 성인 선충의 생식 세포의 파괴로 멸균된다. <ol><li> 알칼리 차아 염소산 용액으로 임신하는 자웅 동체를 처리하여 L1 유충의 동기화 된 인구를 준비 (10 ㎖ 솔루션 : 5M 수산화 나트륨, 가정용 표백제의 2.5 ㎖, 7 ㎖의 H 2 O 0.5 ㎖). 조심스럽게 어른 벌레까지이 솔루션에서 임신하는 자웅 동체 바위의 용해. 계란은 보존되고, 저속 원심 분리에 의해 펠릿 화 될 수있다. </li><li> (3g의 KH 2 PO 4, 6g HPO 4, 5g의 NaCl, 1 ㎖ 1 M 망초, H 2 O L.는 고압 증기 멸균 후 망초로 추가 1 2 NA) M9 버퍼에 계란 준비를 3 회 반복한다. 충분한 산소를 제공하기 위해 밤새 진탕 M9 버퍼와 바위의 5 ML의 최종 볼륨에 15 ㎖의 플라스틱 관에서 계란을 재현 탁. </li><li> L1 유충 이틀 RNAi의 세균을 도금 한 후 OP50, 또는 하루 파종 후 보충 판에 추가해야합니다. 그들은 성인의 단계에 도달 할 때까지 사흘 동안 20 ° C에서 벌레를 품어 나. </li></ol></li><li> 어른 벌레는 자궁에서 개발 태아를 시각화하기 위해 충분히 높은 배율과 해부 현미경을 사용하여 무균 득점 할 수있다. 성공적인 생식 세포의 손실은 계란의 명확한 자궁 무효로 표시됩니다. </li><li> 웜은 또한 고정하고 핵산으로 염색하여 획득 할 수있다생식 세포의 핵의 시각화 및 개발 배아를 용이하게 CID 염료 diamindinophenylindole (DAPI). 빠른 DAPI의 얼룩은 watchglass 9 M9 버퍼의 하락으로 벌레를 선택하여 달성된다. <ol><li> 0.2 NG / m의 1 ㎖와 홍수 시계 유리. DAPI 95 % 에탄올 용액에 약 5 분 동안 앉아 보자. </li><li> 슬라이드에 VectaShield 한 방울로 웜을 선택하고 coverslip을 커버. </li><li> 최적의 염색에 대한 어두운 하룻밤에 4 ° C에서 인감 커버 슬립 및 저장하지만 슬라이드는 즉시 볼 수 있습니다. 존재 또는 UV 램프 및 필터가 장착 된 형광 현미경을 사용하는 생식 세포 핵의 유무에 대한 점수. </li></ol></li></ol> 3. 가스 크로마토 그래피에 의해 지방산 흡수를 확인 C.의 전체 지방산 조성물 엘레은 분리 가스를 사용 CHR 정량화 지방산 메틸 에스테르 (장의 이미지)의 제조에 의해 결정될 수있다omatography 4. <ol><li> 물 접시 오프들을 세척 및 실란 13mm × 100 mm 유리 나사 정상 관에 전송하여 500 ~ 1,000 어른 벌레를 수집합니다. </li><li> 벌레가 중력에 의해 정착하자, 다음 유리 파스퇴르 피펫으로 가능한 한 많은 물을 제거합니다. </li><li> 물에 한 번 벌레를 세척 한 후 다시 가능한 한 많은 물을 제거합니다. </li><li> 장의 이미지 수욕에서 1 시간 동안 70 ° C에서 1 2.5 % H 2 ㎖의 메탄올 SO 4, 다음 열을 추가하여 형성된다. </li><li> 물을 욕조에서 튜브를 제거하고 1 분 동안 냉각. </li><li> 1.5 ㎖의 물 및 헥산 0.25 ㎖를 추가하여 장의 이미지의 압축을 풉니 다. </li><li> 요점을 되풀이 튜브와 적극적으로 흔들어. </li><li> 수성 용매에서 헥산을 분리하는 최대 속도로 1 분 탁상 임상 원심 분리기에서 원심 분리기 튜브. </li><li> 수성 단계 중 하나를 전송하지 않도록주의하고, GC 유리 병 안에 GC 유리 병 삽입에 헥산 (꼭대기 층)를 전송합니다. GC의에 대한(분석), 헥산에 대단한 호평 1-2 μL은 장의 이미지 분석에 적합한 극성 모세관 가스 크로마토 그래피 칼럼에 주입된다. 애질런트 7890 GC 인젝터는 1.4 ㎖ / 분의 유속으로, 250 ° C로 설정되고, GC 오븐은 1 분 동안 유지되는 130 ° C의 초기 온도, 프로그래밍된다. 그 후, 온도는 10 ° C / 분까지 190 ° C를 램핑 된 후 210 ° C까지 5 ° C / 분에서 다시 램핑 및 추가 1 분 동안 유지. </li><li> , DGLA의 흡수가 발생했습니다 확인 C의 식별을위한 본격적인 표준을 사용하여 불꽃 이온화 검출 (FID) 또는 질량 분석기 (MS) 검출에 의해 장의 이미지를 분석하려면 지방산 엘레 간스. </li></ol>

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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+Mediator+subunit,+MDT-15,+integrates+regulation+of+fatty+acid+metabolism+by+NHR-49-dependent+and+-independent+pathways+in+C.+elegans.">A Mediator subunit, MDT-15, integrates regulation of fatty acid metabolism by NHR-49-dependent and -independent pathways in C. elegans.</a> Genes Dev. 20, 1137-1149 (2006).</li><li>Lesa, G. M., 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=Long+chain+polyunsaturated+fatty+acids+are+required+for+efficient+neurotransmission+in+C.+elegans.">Long chain polyunsaturated fatty acids are required for efficient neurotransmission in C. elegans.</a> J. Cell Sci. 116, 4965-4975 (2003).</li><li>Brock, T. J., Browse, J., Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+desaturation+and+the+regulation+of+adiposity+in+Caenorhabditis+elegans.">Fatty acid desaturation and the regulation of adiposity in Caenorhabditis elegans.</a> Genetics. 176, 865-875 (2007).</li><li>Goudeau, J., 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=Fatty+acid+desaturation+links+germ+cell+loss+to+longevity+through+NHR-80/HNF4+in+C.+elegans.">Fatty acid desaturation links germ cell loss to longevity through NHR-80/HNF4 in C. elegans.</a> PLoS Biol. 9, e1000599 (2011).</li><li>Yang, F., 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=An+ARC/Mediator+subunit+required+for+SREBP+control+of+cholesterol+and+lipid+homeostasis.">An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis.</a> Nature. 442, 700-704 (2006).</li></ol>

저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

여기에서 우리는 C의 보충하는 방법을 설명 식이 불포화 지방산의 elegans. 위에서 언급 한 바와 같이 된 PUFA의 이중 결합의 반응 특성이 지방산은 열과 빛 (11)을 통해 산화에 민감하게 반응하는 원인이되기 때문에주의를 PUFA 보충 판의 제조에주의해야합니다. 산화를 방지하기 위해, 용지가 어두운 환경에서 55 ° C 저장 플레이트로 냉각되면, 액체 한천 배지에 PUFA을 추가하는 것...

지방산은 세포막의 필수 구조적 구성 요소뿐만 아니라 효율적인 에너지 저장 분자이다. 또한 지방산은 리파제에 의해 세포막에서 절단 될 수 있고, 효소 ​​적 시그널링 이펙터 하나를 생성하도록 변형 될 수있다. 자연적으로 발생하는 고도 불포화 지방산 (PUFAs에)는 두 개 이상의 시스 이중 결합을 포함하고 있습니다. 오메가 -3 지방산과 오메가 -6 지방산은 지방산의 메틸 말단에 대하여 이중 결합의 위치에 기초하여 서로 구별된다. 건강한 식단은 오메가 -6와 오메가 -3 지방산을 모두 필요로한다. 그러나, 서양 식단은 오메가 -6 지방산과 오메가 -3 지방산이 가난한 특히 풍부하다. 높은 오메가 -6 오메가 -3 지방산 비율은 심장 혈관 및 염증성 질환의 위험이 증가하지만, 특정 지방산의 정확한 유익하고 해로운 기능이 잘 이해되지와 연관된다. 거위 Caenorhabditis의의 ELEGA그것은 오메가 -3 불포화, 대부분의 동물 3,4 부재 활동 등 오메가 -6와 오메가 -3 지방산의 범위를 생성하는 데 필요한 효소를 모두 합성 때문에 NS 지방산 기능 연구에 유용하다. 지방산 불포화 화 효소 결핍 돌연변이 발생 및 신경 학적 결함 4-6의 범위에 이르는, 특정 된 PUFA를 생산하지 못한다. 식이 지방산의 생리 학적 효과를 연구하기 위해, 우리는 C.에 돌연변이 및 RNAi의 노크 다운 기술을 모두 사용하여 유전자 분석과 호환되는 생화학 적 분석을 개발했다 엘레. 특정 된 PUFA와 보충제는 이전에 붓는 한천 배지에 지방산 나트륨 염 용액을 첨가함으로써 달성된다. 이는 E. 의해 PUFA 흡수 초래 이 세균의 세포막에 축적 대장균 음식 소스. C. 엘레는 PUFA 함유 박테리아를 섭취하고,이식이 보충제으로 결함을 구출하기에 충분하다PUFA가 결핍 된 돌연변이 체의 TS. 대부분 지방산 보충제 그러나 특정 오메가 -6 지방산, 특히 디 호모-γ 리놀렌산 (DGLA, 20시 3분 N-6) C.의 영구 파괴 원인을 야생형 동물에 아무런 악영향이 없다 엘레 배아 세포 7,8. 기체 크로마토 그래피는 세균성 음식 소스 (OP50 HT115 또는 하나)에서뿐만 아니라, 선충의 보충 지방산의 흡수를 모니터링하기 위해 사용된다. 미디어 세제 TERGITOL (NP-40)의 첨가는 전체 플레이트와 E. 의한 지방산의 더 효율적인 흡수를 통한 지방산의 균일 한 분포를 허용 대장균 및 선충. 우리는 불포화 지방산이 쉽게 박테리아와 C.에 의해 흡수되는 것을 발견했다 엘레 있지만, 포화 지방산의 흡수는 훨씬 덜 효율적이다. 이 문서에서는 지방산과 한천 매체를 보완하는 방법을 단계별로 설명합니다,뿐만 아니라 일의 지방산 흡수를 모니터링하는 방법과 같은 것가스 크로마토 그래피를 사용하여 전자 선충.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

지방산은 다수의 세포 기능에 필수적이다. 이들은 세포막의 소수성 코어를 구성하고, 다양한 신호 전달 경로에 참여하는 등 효율적인 에너지 저장 분자를 봉사. Caenorhabditis의 엘레 오메가 -6와 오메가 -3 지방산의 범위를 생산하는 데 필요한 효소를 모두 합성한다. 이것은 간단한 해부학 가능한 유전 도구의 범위와 함께, 그것은 지방산의 기능을 연구하는 매력적인 모델합니다. 식물 지방산의 생리 학적 효과를 매개하는 유전자 전달 경로를 조사하기 위해, 우리는 C.를 보완하는 방법을 개발했다 불포화 지방산 elegans의 다이어트. 보충제 웜의 지방산 조성을 변경하는 효과적인 수단이며, 또한 지방산 결핍 돌연변이 체의 결함을 구하기 위해 사용될 수있다. 우리의 방법은 지방 acidsodium 소금으로 보충 선충의 성장 매체 한천 (NGM)를 사용합니다. 보충 판에있는 지방산은 incorpora가테드 다음 C.에 의해 흡수되어 세균의 음식 소스의 세포막에 보충 박테리아에 먹이 엘레. 또한 보충 웜 발생 지방산 조성의 변화를 모니터링하기 위해 가스 크로마토 그래피 프로토콜을 설명한다. 이것은 C의 크고 작은 집단의 다이어트를 보완 할 수있는 효율적인 방법입니다 엘레,이 방법에 대한 응용 프로그램의 범위에 대한 허용.

C의 보충 엘레 다이어트는 세균의 막에 지방산 통풍 관과 통합하는 세균의 먹이 원의 능력에 의해 제한된다. E.의 능력을 결정하기 위해 그 멤브레인으로 각종 지방산을 동화 대장균 OP50은 OP50가 없음 서플리먼트, 0.1 ㎜ 및 스테아린산 (18시), 올레산 나트륨 (18시 1분 N-9), 및 소듐 DGLA (20 0.3 mM의 농도와 함께 매체 상으로 도금 하였다 : 내지 3n-6). 플레이트를 어두운 곳에서 ?...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

&lt;em&gt; 사용 Caenorhabditis의의 우 •은 &lt;/ EM은&gt; 개발 및 생리학에 고도 불포화 지방산의 기능을 탐구 할 수있는 유용한 모델입니다. 이 프로토콜의 &lt;em&gt; C.의 보충의 효율적인 방법을 개시 엘레 &lt;/ EM&gt; 고도 불포화 지방산 다이어트.

에있는 고도 불포화 지방산의식이 보충제<em&gt; 예쁜 꼬마 선충</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K 조회수.  Washington State University.  사용 Caenorhabditis의의 우 •은 개발 및 생리학에 고도 불포화 지방산의 기능을 탐구 할 수있는 유용한 모델입니다. 이 프로토콜의 C.의 보충의 효율적인 방법을 개시 엘레 고도 불포화 지방산 다이어트. 

비디오: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

분리 및 체외에서</em의> 활성화 Caenorhabditis 엘레간스 정자

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

연구

생물학

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

솔리드 플레이트 기반식이 제한에 Caenorhabditis 엘레간스</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

에서 프로스타글란딘 추출 및 분석<em&gt; Caenorhabditis의 엘레</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

의 재배<em&gt; 예쁜 꼬마 선충</em&gt; 실험실에서 세 가지 차원에서

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

그대로 꼬마 선 충 에 산소 소비 속도의 측정

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

예쁜꼬마선충의 정화 및 분석 세포외 소포

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Caenorhabditis 예인단에서 조직 특정 전립선 성 감소를 정량화

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegans의 집계 정량화 자동화

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...