이 작품은 국립 과학 재단 (FT에 MCB-0923794)에서 교부금에 의해 지원되었다.

1. Perithecia 생산 <ol><li> F.의 비옥한 변형율과 6.0 cm의 diam 배양 접시에있는 센터 접종 당근 한천 10 graminearum은 (PH-1을 추천). </li><li> 장소는 실내 온도 (18-24 ° C)에서 밝은 형광등 아래에서 요리를 주사하고 균사체가 배양 접시 (3-5일)의 바깥쪽 가장자리에 도달하기 전까지는 성장할 수 있습니다. 상업용 가정용 형광등은 fruiting 기관 형성 및 방전을 자극 맞게 설정되어 작동합니다. </li><li> 부드럽게 멸균 이쑤시개로 공중 mycelia를 제거합니다. </li><li> 멸균 유리 hockeystick 또는 멸균 유리 막대에 둥근 엔드와 표면에 1.0 ML 2.5 % 십대 초반 60 AQ를 배포합니다. </li><li> 불빛에 번호판을 반환합니다. 접시에 Parafilm를 추가하지 마십시오. </li><li> 24 시간 후, 접시의 표면이 반짝 이는 모습이 있어야합니다. 만약 mycelia 다시 나타납니다, 자국 표면과 다시 신청 십대 초반-60 솔루션입니다. </li><li> 다음 주 동안 perithecia 개발을 관찰. 의 중간 단계perithecium 개발은 부드럽게 RNA 추출 (그림 1)에 대한 냉동 한천과 플래시의 표면은 부서에서 수확한 수 있습니다. </li><li> 성숙한 포자는 7 일 의한 접시의 뚜껑에 축적됩니다. 처음에는 이러한 것들이 단지 해부 현미경으로 볼 수 있지만, 눈에도 보일 할만큼 일 9 그들은 충분한 농도로 축적됩니다. </li></ol> 2. Ascospore 방전 분석 <ol><li> 일 6 십대 초반 솔루션의 응용 프로그램이 후 나무 보어와 한천의 1cm의 diam 원 그만. 또는 메스는 유사한 크기의 세그먼트를 제거하는 데 사용할 수 있습니다. 동그라미는 반으​​로 썰어 각각의 절반이 유리 현미경 슬라이드에 둘 수 있습니다. </li><li> 동양이 조각 fruiting 기관을 포함하는 표면이 슬라이드의 표면에 직각 수 있도록하고, 포자는 슬라이드의 길이를 아래로 해고되었습니다. </li><li> 조명 아래에서 하룻밤 습도 챔버에서 슬라이드를 놓습니다. 포자는 계속 축적됩니다슬라이드와 육안 다음날 아침 (그림 2) 볼 수 있습니다. </li><li> 축적된 포자는 물로 씻어 슬라이드 및 원하는 경우 계량 수 있습니다. </li><li> 잠재 ascospore 방전 억제제는 분석의 조립시 한천 블록의 뒷면에 추가하여 assayed 수 있습니다. 배출을 줄이고 일부는 이온 채널 억제제는 이전에이 기술을 15 assayed되었습니다. </li></ol> 3. 재조합 자손의 세대 <ol><li> F.의 두 ​​변종과 당근 한천을 inoculating하여 십자가를 개시 graminearum (한 알 + 한 알 -), 배양 접시의 반대편에 접종 포인트를 배치. </li><li> 일단 균사가 표면을 가득했습니다 공중 부분을 긁어서하고 위에서 설명한 십대 초반-60 솔루션을 적용합니다. 균사 만나는 배양 접시의 측면에 유의하도록 마커를 사용합니다. </li><li> 불빛에 문화를 반환하고 perithecia가 개발한 때까지 알을 품다, 그리고 cirrhi 수있다총이 두 문화 사이의 교차로를 따라 perithecia에서 형성합니다. </li><li> 두 문화의 교차로 따라 perithecia을 볼로 해부 현미경에서 가져. 곤충 핀을 이용하여 소독 및 멸균 물에 담근, 핀의 끝부분에 살짝이 테두리를 따라 cirrhus 만지지. 핀에 수분이 cirrhus이 핀에 집착해서는 안된다. </li><li> 포자 씻어하기 Eppendorf 튜브에 200 μl 멸균 물 위에 cirrhus로 핀의 팁을 씻어. </li><li> 화염 핀, 다시 그것을 축축하게하다 다른 cirrhus를 선택하십시오. 10-15 cirrhi를 선택하고, 물을 별도의 튜브에 각각 배치합니다. </li><li> 간단히 와동 튜브는 cirrhi를 일시 중지 포함하는. </li><li> 9cm 페트리 접시에 MMTS 매체 1 피펫과 장소와 포자 현탁액의 60 μl를 제거합니다. 멸균 하키 스틱으로 퍼졌다. 나머지 포자 서스펜션은 최대 일주일 동안 4 ° C에 보관하고 필요한 경우 replated 수 있습니다. </li><li> Incuba3~5일을위한 실내 온도 (필요하지 빛)에서 테는 아주 작은 식민지는 (그림 3) 나타날 때까지. 식민지 간의 phenotypes 두 종류가있을 것이다. 알 - 식민지는 알 +는 식민지보다 sparser의 균사가됩니다. 재조합 perithecia에서 Cirrhi는 대략 동등한 비율의 두 식민지의 ph​​enotypes을 보여줍니다. 그들을 단지 하나의 표현형의 식민지로 번호판을 폐기하십시오. 때로는 두 개 이상의 phenotypes 다른 요인의 분리로 인해 발생합니다. </li><li> V8 또는 저장 당근 한천에 식민지를 이동합니다. 올바른 표현형을 확인하기 위해, Czapek의 Dox 한천 (알 - 변종이 Czapek의 Dox에 성장하지 않습니다)에서와 염소 산염 (알 이상의 변종이 염소 산염과 PDA에 성장하지 않습니다 0.5 M 칼륨 염소 산염)와 PDA에서 테스트 식민지. 원하는 자질에 대해이 식민지를 검사합니다. </li></ol> 4. 대표 결과 perithecia 생산 목표의 잔디가하는 것입니다어떤 mycelia 또는 명백한 포자없이 perithecia. 문화의 표면은 검은색 벨벳 (그림 1)와 유사한 표시됩니다. 24 시간에서 십대 초반의 적용 후 한천의 표면은 약간의 광휘가 있어야합니다. mycelia 다시 표시면 판은 perithecium 개발을 유도 정도 까진되지 않았을 수 있습니다. Ascospore 방전 항상 야생 형 변형에서 분석 한천의 여러 단위로 제공하고 있습니다. 그 화재하지 않으면, 그때도 당신의 perithecia 너무 어린이나 너무 많은 나이 잖아. 그들은 너무 나이가있다면 여러 균사가 그것을 약간 흰 색조를주는 문화의 표면 위에 나타납니다. 이런 경우가 아닌 경우, 그들은 너무 어려 될 수 있으며, 검정은 24 시간 후에 다시 시도해야합니다. 때때로, 문화가 잘 배출되지 않으며, 실험 반복해야한다. 당신은 적절한 조명 조건 하에서 분석을 수행하십시오. 재조합 자손 <p class="jove_content"&gt; 재조합 perithecia는 두 변종 사이의 교차로를 따라 perithecia의 대부분을 구성해야합니다. 우리는 보통 십자가에서 10 cirrhi을 처리하고 그 중 최소한 4-6은 재조합해야합니다. 아주 가끔씩, 돌연변이 짝짓기의 변형을 방지합니다. 부모 중 하나가 비정상적 성장 표현형를 가지고있다면, 2 개 이상의 phenotypes는 MMTS의 식민지들 사이에서 존재할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3895/3895fig1.jpg" /> 그림 1. 당근 한천에 perithecium 개발의 단계. 십대 초반의 적용 후 왼쪽, 72 H. 붉은 안료와 표면에 검은 알갱이처럼 보이는 매우 젊은 perithecia를 적어 둡니다. 맞아요, 144 H에서 성숙의 perithecia가 형성되고있다. 두 문화의 천박한 mycelia의 거의 완전한 부재를 적어 둡니다. <img alt="그림 2" src="/files/ftp_upload/3895/3895fig2.jpg" /> 그림 2. 방전 분석. 주황색 당근 한천 플러그 지향하므로 THA입니다강제 표면 성숙 perithecia에서 배출 t 포자는 유리 슬라이드의 표면에 축적하실 수 있습니다. 15 시간 축적 시간. <img alt="그림 3" src="/files/ftp_upload/3895/3895fig3.jpg" /> 그림 3. 알 + 및 선택적 MMTS 매체에서 알 - 식민지 사이에 성장의 차이. 알 - 식민지는 (화살촉) 작은 수 있습니다. 바보 + 식민지는 (화살표) 강력한 성장을 보여줍니다. 사진은 7 일 후에 촬영.

<ol>
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우리는 공개 할게 없다.

F. graminearum은 특히 잘 주석 게놈 (의 가용성으로 인해 fruiting 기관 개발 연구에 적응되어 <a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB /</a> 4), 그리고의 가용성 Affymetrix- microarray 6 기반. 이것은 ascospore 방전 7,11,3위한 중요한 유전자를 식별하는 능력을 촉진하고있다. 여기에 제시된 방법은 연구자가 F.의 fruiting 기관의 유전자 분석을 위해 발달 단계와 함수의 이산 집합에 집중할 수있게 graminearum. 메소드는 또한 문화의 열매에 시달릴 수있는 관련 곰팡이에 쉽게 적용할 수 있으며, 다른 곰팡이 fruiting 본문 유형의 다양한 개발을위한 표준으로 사용할 수 있습니다. 포자 발사 표현형을 평가하는 능력은 포자가 볼 작고 어려운 수종 등에 미묘한 수 <eM&gt; F. graminearum. 깨끗한 슬라이드에 포자의 컬렉션은 포자가 슬라이드를 씻어하고 quantitated 수 있으므로, 시각 평가와 양적 평가 모두를 용이하게합니다. 일부 곰팡이 종들은 포자와 포자가 떨어져 씻어서 수 없습니다을 둘러싸고 점액을 생산하지만, 그들이 슬라이드에 남아으로 미세하게 계산되어야합니다. 우리는 Sclerotinia sclerotiorum의 사실이 발견되었습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Fusarium graminearum 변형 PH-1 </td><td> 곰팡이 유전학 증권 센터, 캔자스 </td><td> FGSC 9075 </td></tr><tr><td> 십대 초반 60 </td><td> 시그마 - 올드 리치 </td><td> P1629 </td></tr><tr><td> Czapek의 Dox 한천 </td><td> Difco, Becton 디킨슨 </td><td> 233,810 </td></tr><tr><td> 칼륨 염소 산염 </td><td> 시그마 - 올드 리치 </td><td> 255,572 </td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

Fusarium graminearum은 filamentous 곰팡이의 개발과 pathogenicity에 공부를 모델 시스템이되었습니다. F.는 graminearum 가장 쉽게 당근 한천에 perithecia라는 fruiting기구를 생산하고 있습니다. Perithecia는 perithecium 개발의 특정 단계에서 생산되는 다양한 조직 유형을 포함합니다. 이들은 (외모 순서) perithecium의 이니셜 (ascogenous 균사를 야기할있는), 외벽, paraphyses (asci 개발까지 perithecium의 중심을 차지하고 멸균 mycelia), asci 및 ascospores 형성 포함 asci 14 이내. 이러한 조직 각각의 개발은 약 24 시간으로 구분되며, 성적 발달 12,8 동안 transcriptomic 연구의 기초가되었습니다. 각 단계의 사진을 포함한 개발의 더욱 철저한 설명을 위해 Hallen 외. (2007)을 참조하십시오. 여기서는 동기를 생성하고 수확을위한 방법을 제시단조는 유전자 조절, 개발 및 생리적 과정의 시간적 연구 perithecia의 잔디를 개발. 이러한 방법은 F. 함께 사용될 구체적으로 작성하고 있지만 graminearum, 기법 fruiting는 문화에 시달릴 수 있도록 제공, 다른 곰팡이의 다양한 사용과 개발에 대한 몇 가지 synchrony가 가능합니다. 우리는 최근 F.의 성적 발달을 연구하기 위해이 프로토콜을 적응했습니다 verticillioides. perithecia을 개발 잔디를 유도하지 못했기 때문에 개별 perithecia는,이 수종에 손을 포착해야하지만, 과정 개발 (Sikhakolli 및 트레일, 게시되지 않은)를 공부를 위해 일했습니다. 곰팡이 fruiting 기관의 가장 중요한 기능은 포자의 분산이다. Ascomycota (자낭은 곰팡이를 생산)의 종의 다양한에서 포자가 통해 (그리고 epiplasmic 유체) 포자의 방출을 유도, 자낭 내에 turgor 압력의 생성으로 인해, 자낭에서 촬영자낭 팁 2,7에서 기공. 강제 ascospore 방전 우리의 연구는 우리가 그 과정에서 변이를 위해 구분할 때 사용하는 &quot;포자 방출 분석&quot;의 개발 결과로했습니다. 여기에서 우리는이 분석의 세부 사항을 제시. F. graminearum는 homothallic이며, 따라서 호환 파트너의 부재에 fruiting기구를 형성할 수 있습니다. homothallism의 장점은 그 교차로는 고유한 특징이 종 14,7의 성욕 발달의 연구를 촉진하고있다 패싯에 대한 자손의 homozygous를 생성할 필요가 없습니다입니다. 그러나 heterothallic 변종은 5,9를 건너 사용할 수있는 생성되었습니다. 그것은 하나의 변형 1의 몇 가지 유전자에 대한 돌연변이를 얻기 위해 homothallic 변종을 통과하는 것도 가능합니다. 이것은 2 계통 하나의 페트리 접시를 coinoculating 통해 이루어집니다. 미팅 포인트 더불어, perithecia의 대다수는 (부모 변종 중 하나에 돌연변이를 억제 제공하지 않습니다 재조합 될 것입니다) outcrossing. perithecia 시대로서, 그들은 강제로 그들을 배출하는 대신 ascospores 실내 masse을 스며 나오게하다. 결과 포자의 exudate은 (cirrhus 함) perithecium의 일각에 앉아 쉽게 개별적인 포자의 복구를 위해 제거할 수 있습니다. 여기 재조합 perithecia 및 재조합 자손의 회복의 식별을 용이하게하기위한 프로토콜을 제시한다.

Watch this Scientific Journal Video about Sexual Development and Ascospore Discharge in Fusarium graminearum at JoVE.com

Sexual Development and Ascospore Discharge in Fusarium graminearum

성 십자가와 재조합 자손의 고립은 사상균에 대한 중요한 연구 도구입니다<em&gt; Fusarium graminearum</em&gt;, 필요한 기술이 성공적으로 이러한 프로세스에 나타난 수행.

의 성적 개발 및 Ascospore 방전 Fusarium graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

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Biology

Sexual Development and Ascospore Discharge in Fusarium graminearum

21.7K 조회수.  Michigan State University. 성 십자가와 재조합 자손의 고립은 사상균에 대한 중요한 연구 도구입니다 Fusarium graminearum, 필요한 기술이 성공적으로 이러한 프로세스에 나타난 수행.

비디오: Sexual Development and Ascospore Discharge in Fusarium graminearum

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Microarray 실험에 대한 Gibberella zeae Ascospore 생산 및 수집.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

연구

생물학

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

IVIS 스펙트럼을 사용하여 내유 종양의 생체내 이미징에서 발광 생물

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

에 잔 심장을 떠올리 Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

인간 배아 줄기 세포를 동결 융해

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

에서 독소 유도와 단백질 추출 Fusarium 종.</emProteomic 연구> 문화

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

마우스 모델에서 유선 개발 및 기능 평가

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

결합 된 DNA-RNA 형광<em&gt; 현장에서</em&gt; 하이브리드 화 (물고기) 차별화 된 여성 마우스 배아 줄기 세포에서 X 염색체의 불 활성화를 연구하는

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

분열 효모 성적 라이프 사이클의 현미경

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

G 단백질 선택적 G​​PCR의 입체는 라이브 셀 서스펜션 형광 분석에서 FRET 센서를 사용하여 측정

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...