Najie 주홍 및 Liqun 바이는이 작품에 동일하게 기여했다. 이 작품은 재정적으로 공공 복지에서 임업 과학 연구를위한 특별 기금 (201,204,501)와 중국 국가 자연 과학 재단 (31170604, 31270688 및 31570638)에 의해 지원되었다. 우리는 영어 쓰기에 자신의 도움이 제안 박사 Holighaus 리트 박사 다니엘 Hickford 감사합니다.

버진 성인 선충 1. 취득 <ol><li> 파인 우드 선충의 분리 및 배양 조건 B를 xylophilus Nxy61 원래 중국 절강 성 닝보 지역에서 병에 걸린 Pinus의 massoniana에서 추출 분리 : 주. <ol><li> (PDA 재료의 표 참조) 감자 포도당 한천을 준비 보 트리 티스 시네 레아를 성장 판. </li><li> 접종 들어 B. 시네 메트에서 10mm의 직경을 갖는 매트 진균의 조각을 펀칭하고 새로운 PDA 플레이트의 중간에 넣어. 문화 어둠 속에서 25 ° C에서 5 D에 대한 B. 시네. </li><li> 문화 어둠 속에서 25 ° C에서 B. 시네을 포함하는 준비된 배양 접시에있는 파인 우드 선충. </li></ol></li><li> B. xylophilus 계란의 취득 <ol><li> 깔때기 아래에 고정 고무 튜브를 놓고 조립하는 랙에 제조 된 깔때기를 개최Baermann 깔때기 장치. 깔때기의 입에 종이 필터 대신 거즈 두 레이어를 놓습니다. </li><li> 유입 경로 설정에 곰팡이 문화를 전송하고 곰팡이 매트가 침지 될 때까지 물을 추가합니다. 2 시간을 기다린 선충은 곰팡이 문화에서 나올 여과지를 통과하고 Baermann 깔때기의 가장 낮은 부분에 정착 것입니다. </li><li> 15 ML의 튜브에 튜브의 바닥에서 물 방울의 첫 번째 부부를 수집하고 천천히 튜브 클램프를 풀어 선충를 수집합니다. </li><li> 실온에서 2 분 동안 3,000 XG에서 위의 컬렉션을 원심 분리기. </li><li> 피펫 물의 상층을 제거하고 멸균 이중 증류 (DD)의 1 mL의 H 2 O와 함께 재현 탁 </li><li> 피펫으로, 새로운 유리 페트리 접시 (6cm 직경)에 위의 부유를 전송합니다. 선충 자유롭게 수영을 할 수 있는지 확인하기 위해 페트리 접시에 무균 DD의 H 2 O 3 ML을 추가합니다. </li><li> 한 유리의 선충을 유지어둠 속에서 25 ° C에서 10 분 동안 배양 접시에요. 계란은 임신 한 여성에 의해 배치됩니다. 때문에 당 단백질 표면의, 계란 유리 페트리 접시의 바닥에 충실 할 것이다. </li><li> 계란을 방해하지 않고, 조심스럽게 물과 벌레를 제거합니다. 건조에서 계란을 방지하기 위해 즉시 유리 페트리 접시에 무균 DD의 H 2 O 3 ML을 추가합니다. </li><li> 단계를 반복 1.2.8 배는 모든 애벌레와 어른을 제거하고 순수한 계란을 얻었다. 페트리 접시에 무균 DD의 H 2 O의 추가 2 ㎖ (총 5 ㎖)를 추가합니다. </li></ol></li><li> 네 번째 단계 청소년의 취득 (J4 유충) <ol><li> 해치 25 ℃에서 어둠 속에서 24 시간 동안 수집 된 계란 J2 유충을 얻었다. </li><li> 2 분 동안 3,000 XG에 15 ML의 튜브, 원심 분리기에 피펫으로 J2 유충을 전송, 물 상층을 제거합니다. </li><li> 멸균 DD의 H 2 O의 200 μL와 J2 유충을 재현 탁하고로 전송B. 시네을 포함하는 PDA 판. </li><li> 문화 어둠 속에서 25 ° C에서 52 시간의 J2 애벌레. 이 시점에서, 대부분의 유충 J2 J4 유충의 초기에 개발 된 것이다. </li></ol></li><li> 처녀 성인 선충의 취득 <ol><li> 알코올 버너 동안 가열 한 후 유리 모세관을 그려 선충을 따기위한 바늘을 준비합니다. 주 :이 모세관 바늘의 팁은 일반적으로는 단일 선충류를 픽업하기위한 도구로서 이용 될 수 있도록, 직경 50㎛의 성인 선충 대략 차체 폭 할 필요가있다. </li><li> Baermann 깔때기를 조립하고 단계 1.2.1에 기술 된 바와 같이, 2 시간 동안 곰팡이 문화 선충을 추출 - 1.2.3. </li><li> 깨끗한 페트리 접시에 물을 J4을 전송합니다. </li><li> 실체 현미경 하에서 선충을 관찰하고 마미야와 기요하라 (8) (그림 1에서 설명 된 바와 같이, 성기 형태로 남성과 여성을 구분 </strong&gt;). 제조 된 모세관 바늘로 원하는 섹스의 선충을 선택하고 남성 또는 여성에 대한 적절한 진균 배양 접시에 전송합니다. 주 : 남성보다 여성이 60에서 여성의 비율에있다 - 70 %. 여성 외음부는 남성 spicule에서 분명히 다르다. 성인에 비해 초기 단계 J4에서 spicule 또는 외음부는 작지만 여전히 쉽게 볼과 구별 될 수있다. 선충은 항상 때로는 남성과 여성을 구별에 어려움을 증가 물에서 움직이고있다. </li><li> 문화 어둠 속에서 25 ° C에서 또 다른 24 시간의 J4 애벌레. 참고 : 모든 선충은 성인으로 발전해야하지만, 그들이 결합 해제하기 때문에, 그들은 처녀 성인으로 간주됩니다. </li><li> Baermann 유입 경로 둘을 조립하고 단계 1.2.1에 기술 된 바와 같이, 2 시간 동안 곰팡이 문화 처녀 성인을 추출 - 1.2.3, 그리고 두 개의 분리 된 깨끗한 페트리 접시에 처녀 성인 남성과 여성을 전송합니다. </li></oL&gt; </li></ol> 2. 관측 및 짝짓기 행동의 비디오 녹화 <ol><li> 여러 오목 슬라이드를 준비하고 자신의 우물 각 멸균 DD의 H 2 O의 200 μL를 추가합니다. </li><li> 모세관 바늘로 준비된 처녀 성인 남성 또는과 여성을 선택하고 각 오목 슬라이드의 물에 하나의 선충을 넣어. </li><li> 그들이 새로운 수성 환경에 적응할 수 있도록 1 시간 동안 잘 내부의 처녀 성인을 유지합니다. </li><li> 피펫을 사용하여 다른 잘 하나에서 둘 중 하나를 전송하여 처녀 남성과 여성의 쌍. </li><li> 실체 현미경 아래 오목 슬라이드에 1♀ + 1♂ 조합의 짝짓기 행동을 관찰한다. </li><li> 버진 성인의 결합 효율을 분석하기 위해, 그 결합 성공률 (R)을 계산하고, 무작위로 선택된 성인 쌍의 것과 비교한다. 교미가 공부 복제의 총 개수에 비해 관찰 된 복제의 백분율 R을 계산한다. </li><li&gt; 결합 선택과 intrasexual 경쟁, 그룹을 조사하고 두 개의 서로 다른 조합으로 처녀 선충을 준수합니다 : 1♀ + 3♂과 + 1♂ 3♀합니다. </li><li> 정량적 분석을위한 데이터를 수집하기 위해, 비디오 현미경을 사용하여 결합 동작을 촬영. 일반적으로, 그들이 교미 후 멀리 이동할 때 선충이 모멘트에 오목 슬라이드의 웰에 배치 된 시점으로부터 전체 정합 프로세스는 2 시간 이내에 완료된다. 따라서 하나의 복제에 적어도 2 시간 동안 연속하여 정합 처리를 기록한다. 최소 30 전체는 각 조합의 총에 복제합니다. 주 : 기록 과정에서 증발을 보상 할뿐만 더 무균 DD의 H마다 0.5 H 2 O (통상 50 μL)을 추가한다. </li></ol> 3. 데이터 수집 <ol><li> 컴퓨터 화면에 신중하게 모든 동영상을보고 성적 하위 행동 특성. </li><li> 결합하여 일의 기간을 측정비디오 레코더의 전자 프레임 별 기능. 수동 종단점으로부터 시작 시간을 뺀다. </li><li> 1♀ + 1♂ 조합은 각 녹화를 분석하여 다음과 같은 이벤트의 지속 시간을 측정한다 : <ol><li> 검색하는 시간을 측정 (즉, 암수가 그들이 발생하는 모멘트에 오목 슬라이드의 웰에 넣고 서로 접촉 할 때까지의 시간). </li><li> 접촉의 지속 시간을 측정한다 (즉, 수형 정확하게 제 교미 위해 여성의 외음부에 위치하는 순간에 제 접촉까지의 시간). </li><li> 교미의 지속 시간을 측정 (즉, 자신이 사정 한 후 아래로 여성의 몸에서 슬라이드까지 남성이 정확하게 여성의 외음부에 위치 할 때까지의 시간). 참고 : 일부 선충은 2 시간에 여러 번 교접 할 수 있습니다. 이러한 경우, 라스의 끝에서 제 교미 교미 기간을 측정사이의 시간을 포함하여 t 하나. </li><li> 여운의 지속 시간을 측정한다 (즉, 교미 후 기간 암수 수영 서로 떨어져 그리고 더 선충의 몸 길이 이하의 간격으로 분리 될 때까지 (통상 1 ㎜)). </li></ol></li><li> 교미 주파수와 모든 교미 기간과 다른 조합의 각각에 대해 2 시간에있는 모든 교미의 평균 시간을 계산합니다 (즉, 1♀ + 1♂, 3♀ + 1♂ 및 1♀ + 3♂). </li><li> 즉시 끊김없이 수행되는 첫 접촉 후 짝짓기 성공률로 상이한 조합에 대해 먼저 접촉 (P)에 대한 상대 성공율을 계산한다. 접촉 단계에서는 여러 연락처는 실제 교미하기 전에 관찰 될 수 있습니다. <ol><li> 두 개인 사이의 거리는 상기 본체의 길이 이상이면 이상으로 하나의 접촉을 고려한다. 하나의 복제, 기록 P 100 %의 교미의 난에 대한의는 휴식없이 첫 접촉 후 즉시 관찰, 또는 0.00 % 더 교미가 관찰되지 않은 경우 또는 교미하지만 두 개 이상의 접점 후이 있다면. 참고 : 여기에 설명 된 각 조합에 대한 30 복제가 있었다. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

행동 테스트는 화학 생태학, 신경 생물학, 분자 생물학, 유전학의 기본적인 부분입니다. 선충, 특히 C. 엘레은 광범위하게이 분야에서 연구에 사용됩니다. 선충의 짝짓기 행동은 이전에 10-11을 조사하고있다. 그러나, B.의 xylophilus는 자웅 이체와 자웅 동체 C. elegans의 다른이며, 그 결합 과정은 다른 행동 기능 9있다. 최근 Bursaphelenchus의 okinawaensis, ...

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, is one of the most destructive invasive species, causing the wilting and eventual death of pine trees. This pathogenic nematode is believed to be native to the USA, but it was transported to several countries, including China, Japan, South Korea, and Portugal. Recently, it has also been reported in Spain, killing millions of pine trees at huge economic losses and threatening forest production and ecological stability1-5.
Once a host pine tree is infected by pinewood nematodes, thousands of millions of offspring are propagated rapidly within the trunk. This causes xylem dysfunction, resulting in wilting and eventually in the death of the host tree6. However, there is currently no efficient way to control this disease. Mating behavior might play an important role in the high fecundity of this nematode7. We therefore investigated the mating behavior of B. xylophilus in the laboratory, trying to find an effective way of disturbing its mating and decreasing its fecundity. 
This protocol is intended to introduce the detailed methods of how to obtain the virgin adults of B. xylophilus and how to observe and analyze the mating behavior using a video recorder and a stereomicroscope. This protocol can also be used as a reference for behavioral studies of other nematodes.

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			<td height="18" style="height:18px;width:233px;">B. xylophilus isolate Nxy61</td>
			<td style="width:123px;"></td>
			<td style="width:125px;"></td>
			<td style="width:463px;">Extracted from Pinus massoniana in Ningbo area of China</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grey mold strain</td>
			<td></td>
			<td></td>
			<td>Obtained from Forestry Academy of China</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Baermann funnels</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">falcon tubes</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">centrifuge</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipette</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sterile dd H2O</td>
			<td></td>
			<td></td>
			<td>Sterilized by high pressure of steam at 121&#176;C for 30 min.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDA medium</td>
			<td>Huankai Bio.</td>
			<td>021050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">incubator</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminar flow</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass capillary</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereoscope</td>
			<td>Leica</td>
			<td>LED5000 RL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted stereo microscope</td>
			<td>Zeiss</td>
			<td>A1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">concave slides</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

observation and quantification of mating behavior in the pinewood nematode, bursaphelenchus xylophilus

관찰과 소나무 선충, Bursaphelenchus의 xylophilus의 짝짓기 행동을 정량화하기위한 방법은, 실체 현미경 아래 설립되었다. B. Xylophilus의 결합 효율을 향상시키고 상대 관찰 가능성을 증가시키는, 버진 성인 배양하고, 조사에 사용. 계란은 물에 선충을 유지하고, 여성은 10 분 동안 알을 낳기 수 있도록함으로써 얻을 수 있었다. 2 단째 유생 (J2)는 어둠에서 25 ° C에서 24 시간 동안 난자를 배양하여 동기화하고, 초기 J4 또 다른 52 시간 동안, 잿빛 곰팡이, 보 트리 티스 시네 레아와 J2를 배양함으로써 얻어졌다. 이 시점에서, 가장 J4 선충은 생식기 형태를 사용하여 남성 또는 여성 인 것으로 명확히 구별 될 수있다. 남성과 여성의 J4 수집하고 처녀 성인 선충을받을 24 시간 동안 두 개의 서로 다른 배양 접시에서 분리 배양 하였다. 처녀 남성과 처녀 여성은 와트의 드롭 쌍 하였다오목 슬라이드의 잘 어. 정합 동작은 실체 현미경 비디오 레코더로 촬영했다. 결합 프로세스의 전체 시간은 82.8 ± 3.91 분 (± SE 평균)이고 4 개의 단계로 나눌 수있다 : 검색, 접촉 교미 및 느린. 구간의 평균 분은 각각 21.8 ± 2.0, 28.0 ± 1.9, 25.8 ± 0.7 및 7.2 ± 0.5이었다. 일레븐 서브 행동을 설명했다 :, 순항 접근, 발생, 반원형 누수 방지 공법, 터치, 위치, 부착, 사정을, 정지를 분리하고, 로밍. 한 여성이 3 명의 남성 3 명의 여성과 한 남성과 함께 그룹화 할 때 흥미롭게도, 분명 내 성적 경쟁이 관찰되었다. 이 프로토콜의 B. xylophilus의 결합 거동을 조사에서뿐만 아니라 다른 선충 ethological 연구를위한 기준으로서 작용에서뿐만 아니라, 유용하고 귀중하다.

처녀 성인의 짝짓기 성공률 (R)가 무작위로 선택된 성인에 비해 유의하게 높았다하는 평균 86.67 %이었다 : 13.33 ± 4.65 % (F = 1301.71, DF = 1, P = 0.0001) (그림 2). 이 발견은 B의 xylophilus 무작위로 선택된 성인보다 처녀 성인 짝짓기 행동을 관찰하기 쉽다는 것을 의미한다. 한 남성이 여성 하나와 결합되?...

Watch this Scientific Journal Video about Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus at JoVE.com

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

파인 우드 선충의 관측 및 짝짓기 행동의 정량화,<em&gt; 소나무 재선충</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

observation-quantification-mating-behavior-pinewood-nematode

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JoVE Journal

Biology

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

9.3K 조회수.  Zhejiang Agricultural & Forestry University. A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

비디오: Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

번식과 Tetrad 분리 Chlamydomonas reinhardtii 유전자 분석을위한

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

연구

생물학

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi는 Necromenic 선충류의 Microinjection에 의해 유전자 최저 및 Transgenesis을 중재 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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

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

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

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

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

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

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

드로소필라의 온도 선호도 행동과 Circadian 리듬의 설계 및 분석

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

관측 및 텔로미어의 정량 및 반복 시퀀스 사용하여 형광<em&gt; 현장에서</emPNA 프로브에와&gt; 하이브리드 (FISH)<em&gt; 예쁜 꼬마 선충</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

엔터로이드에서 증식 및 죽은 세포의 정량화

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With a growing global population and limited arable land, controlling PPN infestation is critical for food production. Compounding the challenge of maximizing crop yields are the mounting restrictions on effective pesticides because of a lack of nematode selectivity. Hence, developing new and safe chemical nematicides is vital to food security. In this protocol, the culture and collection of the PPN species Ditylenchus dipsaci are demonstrated. D. dipsaci is both economically damaging and relatively resistant to most modern nematicides. The current work also explains how to use these nematodes in screens for novel small molecule nematicides and reports on data collection and analysis methodologies. The demonstrated pipeline affords a throughput of thousands of compounds per week and can be easily adapted for use with other PPN species such as Pratylenchus penetrans. The techniques described herein can be used to discover new nematicides, which may, in turn, be further developed into highly selective commercial products that safely combat PPNs to help feed an increasingly hungry world.

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

The present protocol describes a reliable and straightforward method for culturing, collecting, and screening Ditylenchus dipsaci.

식물 기생 선충류 Ditylenchus dipsaci 배양 및 스크리닝

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

파인우드 선충류에서의 RNA 간섭의 적용, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.