저자는 E를 공급 6 월의 미들 감사드립니다 faecalis 균주 및 지침 박테리아 문화. C. 엘레은 연구 기반 프로그램의 NIH 사무소 (P40 OD010440)에 의해 자금 지원 CGC에 의해 제공되었다.

선충류 성장 미디어 1) 준비 (NGM) <ol><li> 50 판을 만들기 위해 1.5 g 염화나트륨 8.5 g 초순수 한천과 1 Lflask 1.25 g의 펩톤을 추가합니다. </li><li> 플라스크에 dH보다 2 O의 487.5 ML을 추가합니다. 알루미늄 호일의 조각으로 플라스크의 입구를 혼합 커버 부드럽게 소용돌이. </li><li> 표준 조건 (121 PSI, 120 ° C, 20 분)에서 고압 증기 멸균하여 솔루션을 소독. </li><li> 솔루션 수욕에서 45 ° C로 냉각 할 수 있습니다. </li><li> 5 MG / ML 주식 솔루션 (에탄올에 용해), 500 μL 1 M 염화칼슘, 500 μL 1 M 황산 마그네슘, 12.5 ML KPO 4 버퍼 (54.15 g에서 500 μL 콜레스테롤 : 솔루션에 순서대로 다음을 추가 dH보다 2 O 500 ML에있는 KH 2 PO 4 17.8 g K 2 HPO 4)와 같은 18 설명했다. </li><li> 멸균 피펫을 사용하여 각 60mm의 폴리스티렌 페트리 접시에 한천 용액 10ml를 추가합니다. 미디어 실내 온도 overn에서 고화 할 수 있습니다광명. </li></ol> B 국물 E. 2) 준비 대장균 미디어 <ol><li> 다섯 100 ML 문화를 만들기 위해 비커에 Bacto 트립 톤, 2.5 g 효모 추출물, 5.0 g 염화나트륨 5.0 g을 추가합니다. </li><li> 500 ML의 최종 볼륨에 멸균 증류수를 추가합니다. 용질이 용해 될 때까지 교반하면서, 핫 플레이트에 솔루션을 가열한다. </li><li> 다섯 개의 유리 병 (최소 200 ML 볼륨)에 B 국물 100 ㎖를 붓는다. </li><li> 표준 조건 (121 PSI, 120 ° C 20 분)에서 고압 증기 멸균하여 솔루션을 소독. </li><li> B 국물을 사용하기 전에 실온으로 냉각 할 수 있습니다. </li></ol> 3) 시드 C. 엘레 유지 보수 플레이트 <ol><li> NGM 플레이트 1 주일 이상 상온에서 건조 할 수 있도록 한 후, E. 몇 식민지로 한 100 ML B 국물 문화를 접종 대장균 (OP50). </li><li> E. 성장 37 ° C 하룻밤 대장균 문화. </li><li> 피펫 하나, 둘, 방울 (약 10081, L) OP50 문화의 각 NGM 플레이트의 중앙 위에. 플레이트는 일반적으로 뚜껑면을 쌓아됩니다. (시드) 피펫 팅 할 때, 스택의 하단에있는 접시에 문화를 pipetting하여 시작합니다. 각 스택을 당신의 방법을 작동합니다. 그것은 문화가 너무 가까이 접시의 가장자리에 접근하지 않는 것이 중요하기 때문에, 파종 후 판을 움직이지보십시오. </li><li> 시드 플레이트 사용하기 전에 적어도 한 주 동안 상온에서 건조하도록 허용합니다. </li><li> 시드 플레이트는 실온에서 밀폐 된 플라스틱 용기에 (반전) 저장됩니다. </li></ol> 4) C. 유지 엘레 균주 <ol><li> 잘 먹이 C를 유지하려면 엘레 주식은 매 3-4 일 새로운 시드 NGM 플레이트에 세 L4S 또는 임신하는 성인, 어린이, 유아를 이동하는 선택 웜을 사용합니다. </li><li> 매장 플레이트 (반전) 바람직 인큐베이터 안에 15에서 - 22 ° C. 기술 분석에서 문화는 20 ° C. ℃에서 보관 하였다 elegans의 발달은 온도에 따라 달라집니다. maintena로온도가 증가 NCE, 발달 단계 사이의 시간은 줄어 듭니다. </li></ol> 트립신 소이 한천 (TSA) E. 5) 준비 faecalis 문화 미디어 <ol><li> 열판에, 교반하면서 50 판을 만들려면, 플라스크에 멸균 증류수 500 mL를 측정하고 종기에 가져다. </li><li> 플라스크에 가루 TSA 한천 20 g을 솔루션을 용해 할 수 있습니다. </li><li> 표준 조건 (121 PSI, 120 ° C 20 분)에서 고압 증기 멸균하여 솔루션을 소독. </li><li> 솔루션 수욕에서 45 ° C로 냉각 할 수 있습니다. </li><li> 멸균 피펫을 사용하여 각 60mm의 폴리스티렌 페트리 접시에 한천 용액 10ml를 추가합니다. 미디어 하룻밤 실온에서 고화 할 수 있습니다. </li></ol> 트립신 간장 국물 (TSB) E. 6) 준비 faecalis 문화 미디어 <ol><li> 교반하면서 250 문화 튜브를 만들려면 철판에 플라스크 따뜻한에 멸균 증류수 500 mL를 측정합니다. </lI&gt; <li> 플라스크에 TSB 한천 분말 15 g을 분말 용해 할 수 있습니다. </li><li> 표준 조건 (121 PSI, 120 ° C 20 분)에서 고압 증기 멸균하여 솔루션을 소독. </li><li> 무균 5 ML 테스트 튜브에 솔루션을 실내 온도까지 냉각 할 수 있도록, 다음, 피펫 2 ㎖. </li></ol> 7) E.에 대한 스트렙토 마이신과 뇌 심장 주입 (BHI) 미디어의 준비, faecalis 문화 <ol><li> 플라스크에 멸균 증류수 500 mL를 측정하고 열판에, 교반하면서, 종기에 가져다. </li><li> 플라스크에 BHI 배지 26g을 추가하고 솔루션을 용해 할 수 있습니다. </li><li> 표준 조건 (121 PSI, 120 ° C 20 분)에서 고압 증기 멸균하여 솔루션을 소독. </li><li> 솔루션 ° C의 물을 욕조에 45 식히십시오. </li><li> 무균 기술을 사용하여, 10 MG / ML 스트렙토 마이신 원액과 혼합하는 소용돌이의 0.5 ML를 추가합니다. </li><li> 각 60mm의 폴리스티렌 페트리 접시에 솔루션의 10 ML을 추가합니다. </li><l난&gt; 허용 미디어 하룻밤 실온에서 응고합니다. 4에서 보관 플레이트 (반전) ° 사용하기 전에 몇 시간까지 C. </li></ol> 8) E. 유지 faecalis 균주 <ol><li> E.을 유지하기 faecalis 주식은 별도의 트립신 간장 한천 (TSA) 판에 연속 개별 식민지에 멸균 루프를 사용합니다. </li><li> 37 ° C에서 하룻밤 문화를 성장. 몇 주 동안 실온에서 밀폐 된 가방이나 상자에 보관 플레이트 (반전). </li></ol> 9) E. 준비 EIW 분석 실험을위한 faecalis 플레이트 <ol><li> E.의 식민지로 2 ML TSB 문화를 접종 faecalis 37에서 밤새 품어 ° C. </li><li> E.의 피펫 20 μL faecalis의 준비된 BHI - 스트렙토 마이신 플레이트의 중앙 위에 문화, 37 밤새 품어 ° C. </li></ol> 10) E. 준비 EIW 분석 실험 (제어 플레이트)에 대한 대장균 플레이트 <ol><li> 100 ML의 B-동생을 접종E.의 식민지 일 문화 대장균 OP50 37 ° C에서 하룻밤 배양 </li><li> 피펫 20 시드되고 NGM 플레이트의 중앙 위에 OP50 문화의 μL 및 시드 플레이트 하룻밤 실온에서 건조 할 수 있습니다. </li></ol> 벌레 분석 11) 계란 <ol><li> 15을 선택 - 20 개최 - L4 C. 준비된 BHI-연쇄상 - E.에서 박테리아의 잔디밭의 중앙 위에 엘레 간스 (그림 1) faecalis 플레이트. BHI 판에 OP50을 많이 전송하지 않도록주의해야합니다. NGM-OP50 플레이트 컨트롤에 L4S의 같은 번호를 선택합니다. </li><li> 20 ℃에서 40 시간 동안 번호판을 품어 </li><li> 20 % 표백제 솔루션을 준비합니다. 증류수의 적절한 볼륨 상업용 표백제 (6.0 % 차아 염소산 나트륨)을 섞는다. </li><li> 플라스틱 뚜껑에 열 별개의 위치 (웜이나 세균 판)에 표백제 용액 10 μL 방울을 추가합니다. </li><li> 웜 픽을 사용하여 각 표백제 드롭으로 한 웜을 전송합니다. 의 부드럽게 소용돌이 팁웜은 선택의 끝을 (해부 현미경 물방울을보고 확인) 씻어 않았는지 확인하기 위해 표백 액에 선택합니다. </li><li> 벌레가 알을 (그림 2) 추방 오픈 버스트 때까지 표피 약 10 분 또는 분해 할 수 있습니다. 판에서 계란을 전송하지 않도록주의하십시오. </li><li> 해부 현미경 사용, 표백제의 각 드롭 계란을 계산합니다. </li></ol>

<ol>
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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

성공적으로이 분석을 수행에서 가장 중요한 단계는 1) C. 잘 자란 주식을 사용하여 분석 플레이트, 3 엘레, 2) 배양 세균의 한 종류)를 정확하게 식별은 E.에 노출 L4 벌레를 개최 faecalis, 4) E.에 노출 시간을 유지 시간을 표백 모든 시련과 5 일관성 faecalis)는 계란 붕괴를 방지하기 위해 10 분 초과하지 않아야합니다. 이 분석을 위해 건강하?...

Caenorhabditis의 엘레, 현미경, 무료 - 생활 거위는 전통적으로 인간 때문에 투명 해부학, 잘 특성화 개발, 완벽하게 배열 된 게놈, 짧은 세대 시간 및 유전 상동의 발달 및 세포 신호 전달 과정을 연구하는 데 사용되는 모델 생물이다 . 더 최근에, C. 엘레 환경 독성학 및 선천성 면역 10, 11 분야의 모델 생물이되었다. 이러한 자기 비옥 자웅 동체 웜 계란에서 부화 2 ~ 3 일 이내에 성적으로 성숙된다. 수명주기, C. 동안 엘레 성인기에 도달하기 전에, 네 애벌레 단계 (L1-L4)를 통해 전달합니다. 하나의 고립 된 자웅 동체 피크 다산 3 일 안에 평균 300 자식을 생성 할 수 있습니다. 생식 성숙 C.에 엘레 자웅 동체는 수정란이 마련되기 전에 몇 시간 동안 자궁 내에서 유지됩니다.한 번에 자궁에 저장된 계란의 정상적인 수 (피크 생산력 중) 열 사이에 다섯 12. 자궁에 계란의 수는 계란 생산의 속도와 달걀 누워의 속도의 함수이다. 수정란은 외음부 (13)의 개구부 주위에 배치 여섯 외음부 근육의 수축에 의해 자궁에서 추방된다. 자웅 동체 특정 motorneurons (HSN)의 따라서 VC motorneurons 근육 수축에 영향을 미치는 외음부 근육 위에 시냅스와 계란을 낳는 행동 5,7,13,14합니다. 자궁 계란의 추방은 신경과 근육의 조정 활동으로 인해 발생합니다. C.의 실험실 문화 엘레은 일반적으로 비병원성 대장균 OP50의 다이어트에 발생합니다. 자연 환경, C.에게있는 엘레 같은 병원성 박테리아와 같은 음식 소스의 다양한 접촉, 그 잠재적으로 유해 할 수 있습니다. 유해한 물질에 노출되었을 때환경, C. 환경이 더 유리한 될 때까지 elegans의 계란을 유지합니다. 아마이 계란 보존 그들의 자손을 보호하기 위해 노력합니다. 웜 (EIW) 분석, C.이 계란 엘레은 잠재적으로 병원성 세균, 환경에서 발견되는 엔테로 faecalis에 노출되어 있습니다. E.의 병원성 형태에 노출 faecalis는 C의 지속적인 창자 감염, 심지어 사망을 일으킬 수 있습니다 엘레 15. 병원성 세균의 다른 형태의 노출이 계란 유지 16,17에 영향을 미치는 것으로 알려져있다, 그러나 효과는 정량화되지 않았습니다. E.의 또한 약간 병원성의 효과는 변종 faecalis는 계란을 낳는 행동에 즉시 치명적인없는 긴장이 연구되지 않았습니다. EIW 분석 실험 C의 자궁에 남아 계란의 수를 계산 포함 엘레 4. 비록 C. 엘레투명, 자궁에 축적 계란 그대로 동​​물의 정량화하기 어려울 수 있습니다. EIW 분석은 임신하는 성인 C. 표백 포함 시간의 고정 기간 동안 세균에 노출 된 엘레 간스. 표백제 솔루션은 뒤에 계란을 떠나 외부 큐티클을 녹인다. 계란은 보호 껍질의 존재로 인해 표백제의 효과 굴절입니다. 표백 후, 하나는 아주 쉽게 표백에 벌레의 자궁에서 풀어 계란의 수를 계산 할 수 있습니다. 기술 분석은 한 번에 자궁에 계란의 수를 정량화 할 수있는 간단하고 저렴하고 빠른 방법입니다, 따라서 E의 효과를 정량화 계란 유지에 faecalis. 이 분석은 계란 보존에 박테리아, 환경 독소 또는 약물의 다른 종류의 효과를 정량화하는 데 사용할 수 있습니다. 이 분석은 세균의 병원성에 대한 화면으로 사용할 가능성이있다.

<table border="0" cellpadding="0" cellspacing="0" width="2472">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agar, ultrapure</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">10906</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Peptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Tryptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Brain Heart Infusion dehydrated medium</td>
			<td style="width:191px;">Carolina Biological Supply</td>
			<td style="width:119px;">781781</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">C. elegans, N2 strain</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">Cholesterol</td>
			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:119px;">A11470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Culture plates for C. elegans</td>
			<td style="width:191px;">Tritech Research Inc.</td>
			<td style="width:119px;">T3308</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Culture plates for E. faecalis</td>
			<td style="width:191px;">Fisher Scientific-Fisherbrand</td>
			<td style="width:119px;">875713</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">E. coli (OP50)</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">E. faecalis strains</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>provided by J. Middleton. All isolates were confirmed as enterococci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>
			all strains grew at 44.5 &#186;C and were catalase negative and hydrolyzed esculin. A simplified&#160;dichotomous key based on pigmentation and fermentation reactions for six sugars&#160;(arabinose, mannitol, methyl-&#945;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&#160;presumptive identification of all E. faecalis strains (Efs lacks pigmentation and is arabinose, MGP&#160;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&#160;were confirmed using the API 20 STREP system (Biomerieux).
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microscope</td>
			<td style="width:191px;">Motic</td>
			<td style="width:119px;">SMZ 168B</td>
			<td>any microscope with transmitted illumination and 50X magnification should be sufficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Streptomycin sulfate</td>
			<td style="width:191px;">Fisher BioReagents</td>
			<td style="width:119px;">BP910-50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">236950</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Yeast extract</td>
			<td style="width:191px;">Acros</td>
			<td style="width:119px;">61180-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

C. elegans의 달걀 누워 동작은 같은 삼투압 1과 진동이 같은 환경 단서에 의해 영향을받습니다. 음식 C.의 총 부재 엘레은 달걀 부설을 중지하고 자신의 자궁 3 수정란을 유지합니다. 그러나 특히 다른 음식의 근원, 특히 병원성 박테리아와 계란을 낳는 행동에 엔테로 faecalis의 효과는 잘 특성화되어 있지 않습니다. 계란에서 웜 (EIW) 분석이 경우 E.에서 박테리아의 다른 유형의 효과를 정량화 할 수있는 유용한 도구입니다 faecalis, 달걀 누워 동작합니다. EIW 분석 실험 C의 자궁에 남아 계란의 수를 계산 포함 엘레 4. EIW 분석은 단계적 표백, 임신하는 성인 C.를 포함 엘레는 표피를 제거하고 동물에서 유지 계란을 분리합니다. 이전 표백, 웜 박테리아 (또는 환경 큐의 모든 유형에 노출되는) 시간의 고정 기간 동안. 표백 후, 하나는 아주 쉽게 벌레의 자궁 안에 유지 계란의 수를 계산 할 수 있습니다. E. 후 계란 보존이 분석에서 정량화 증가 faecalis 노출을 쉽게 측정 할 수 있습니다. EIW 분석은 잠재적 병원성 세균이나 환경 독소의 존재를 선별하는 데 사용할 수 있습니다 행동 분석이다. 또한, EIW 분석 계란을 낳는 행동과 같은 세로토닌과 아세틸 콜린 5-9과 같은 신경 전달 물질에 의해 조절하기 때문에 신호 전달 물질에 영향을 미치는 의약품에 대한 화면 도구가 될 수 있습니다.

이 분석은 하나의 C. 내에서 유지 계란의 수를 정량화 할 수 E.에 노출 된 후 엘레 faecalis. L4 벌레가 (자신의 외음부, 그림 1 위의 투명한 열린 공간이 특징) E.에 노출 된 연출 성인을 통해 faecalis. E.에 노출 마흔 시간 후 faecalis는 표백을 수행 하였다. 표피가 표백 액의 붕괴로, 계란 (그림 2) 더 명백하게되었다. 유지 계란의 수는 쉽게 ?...

Watch this Scientific Journal Video about Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay at JoVE.com

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

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

계란에서 웜 (EIW) 분석은 달걀 누워 행동을 정량화 할 수있는 유용한 방법입니다. 달걀 부설의 변화는 모델 유기체의 행동 반응 할 수 있습니다<em&gt; Caenorhabditis의 엘레</em&gt;와 같은 병원성 박테리아에 의해 생성 된 것과 같은 잠재적으로 유해한 환경 물질.

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

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

measuring-effects-bacteria-on-c-elegans-behavior-using-an-egg

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

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16.9K 조회수.  Fairleigh Dickinson University. 계란에서 웜 (EIW) 분석은 달걀 누워 행동을 정량화 할 수있는 유용한 방법입니다. 달걀 부설의 변화는 모델 유기체의 행동 반응 할 수 있습니다 Caenorhabditis의 엘레와 같은 병원성 박테리아에 의해 생성 된 것과 같은 잠재적으로 유해한 환경 물질.

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

Generation of Stable Transgenic C. elegans Using Microinjection

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

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

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

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

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Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

측정 Caenorhabditis 엘레간스</em솔리드 미디어> 수명

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

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 ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

시토크롬를 사용 Mitochondrial 호흡 기능의 시각화 C 데이스 / 호박산 탈수소 효소 (COX / SDH)를 두 번 라벨 Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

에서 Hypoxia의 효과를 연구하기 위해 정의된 기체 환경 만들기<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

두개골 Mesenchyme의 셀룰러 행동을 평가하기위한 Explant 분석

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

C. elegans Chemotaxis Assay

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

C. elegans Chemotaxis Assay

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

C. elegans의 주 화성 분석

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

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

항체 염색법에<em&gt; C. 엘레</em&gt; &quot;동결 크래킹 사용&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

대규모 유전자 넉다운에<em&gt; C. 엘레</em&gt; 강력한 기능 상실 표현형을 생성하는 dsRNA를 먹이는 라이브러리 사용

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

허혈 / 재관류에 대한 산소 결핍 - 기아 모델<em&gt; C. 엘레</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...