우리는 낸시 Papalopulu 및 Xenopus의 electroporation과 지원 Boyan Bonev에 감사하고 있습니다. 우리는 또한 중요한 독서, 제레미 그린과 지원 리우 실험실의 도움이 토론과 회원에 대한 존 Wallingford에 대한 마크 Dionne 주셔서 감사합니다. 이 작품은 BBSRC (BB/E013872/1)와 Wellcome 트러스트 (081880/Z/06/Z)에서 KJL 부여하여 자금을했습니다.

부품 1A. 장비 현미경 : 낮은 전력 목적으로 직립 스테레오 - 해부 범위 <ul><li> 전압 / 펄스 발생기 : BTX ECM 830 스퀘어 웨이브 Electroporation 시스템 </li><li> 피펫 풀러 : P - 87 Micropipette 풀러 (셔터 악기 회사, CA) </li><li> 속이는 사람 : 거친, 또는 결합 굵고 좋은가 준비에 따라 다릅니다. </li><li> Micropipette 홀더 : 파인 과학 도구 </li><li> 전극 : 수제 </li><li> Electroporation 챔버 : 수제 </li></ul> L - 모양의 전극 : <ol><li> 고순도의 8cm 0.4 mm의 텅스텐 와이어 (Goodfellow)과 midpoint에 부착을 잘라 접착제를 (우리가 블루 - 압정을 사용)를 사용하여 1ml 주사기. L - 모양에 주사기 및 굽힘 팁의 끝부분에서 노출된 4cm의 텅스텐 와이어를 떠나 끝에서 1cm (그림 1A). </li><li> 팁 이렇게 끝이 길이가 0.​​5 mm를 측정하는 것을 낸다. 티그의 끝은 전극 종착역입니다. </li><li> 주사기에 여분의 텅스텐 와이어 병렬를 실행하고 전극 펄스 발생기를 연결하는 그것을 사용합니다. </li><li> 전극 한 켤레를 만드는 과정을 반복합니다. </li><li> DC 케이블을 통해 사각형 웨이브 펄스 발생기에 전극을 부착합니다. </li></ol> Electroporation 챔버 <ol><li> ~ 5mm 무독성 플라스티신와 90mm 접시의 바닥 선. </li><li> electroporation 미디어와 함께 요리를 입력합니다. </li><li> 사용 5 호 시계 제조인의 집게는 T - 모양 잘 (그림 2) 싶습니다. 오랜 우물 ~ 2mm X 2mm X 10mm와 짧은을 ~ 2mm X 2mm X 5mm를 측정한다. electroporation 요리 세탁해서 활용할 수 있습니다. </li></ol> 부품 1B. 시약 DNA 또는 유료 macromolecules <ul><li> Micropipettes : 1mm 폭 4 &quot;길이 borosilicate 유리 모세관 (WPI, TW100 - F) </li><li> 문화 미디어 : 일반 수륙 양용 미디어 (NAM) </li><li> &gt; 10 X 재고 : 1100 MM NaCl, 20MM KCL, 10 MM 칼슘 (NO 3) 2 • 4H 2 O, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산). <ul><li> 4 ° C.에 압력솥 및 저장 </li><li> 1 X NAM : 0.1 MM NaHCo 3 0.2 MM 나 3 PO 4 10X 주식, 버퍼에서 희석. </li></ul></li><li> Xenopus laevis tadpoles, 단계 28 </li></ul> DNA 준비 : <ol><li> 표준 프로토콜을 사용하여 표현 plasmids를 준비합니다. </li><li> nuclease 무료로 H 2 0 1 μg / μl의 최종 농도로 DNA를 Resuspend. </li></ol> * 우리는 같은 pCS2로 강한 CMV 프로 모터를 포함하는 벡터, 성공 있었 + [16]. 혈통 분석을 위해, 우리는 대개 0.1 μg / μl의 최종 농도에서 녹색 형광 단백질 (GFP pCS2 +)를 인코딩 DNA를 포함합니다. 0.1-3 μg / μl 사이 [DNA의 농도도 테스트했다. 우리는 발견 0.8 μg / μl inefficiently 표시 셀 아래의 농도, DNA의 농도 반면 이상 2 μg / &amp; MU; 난 electroporation 효율을 향상되지 않았]. Morpholino oligonucleotide 준비 : (참고 :. 수법의 살인인 걸 fluoresceinated해야합니다 (3&#39; - carboxyfluorescein 수정)하거나 유료) <ol><li> Resuspend morpholino의 oligonucleotides (수법의 살인인) (Genetools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> nuclease 무료로 H 2 0 2 mm의 농도에서). </li><li> 65 재고 솔루션의 열 나누어지는 ° C 5 분. </li><li> nuclease 무료 물에 0.5 mm의 최종 농도 희석. </li></ol> * 0.1 - 1mM MO 솔루션은 테스트되었습니다. 0.5 MM MO 솔루션은 많은 mesenchymal 세포 electroporation에 충분했다. Micropipettes <ol><li> borosilicate 유리 모세관 (폭 1mm, 4 &quot;긴 WPI 안돼. TW100 - F)에서 micropipettes를 준비합니다. 8-12밀리미터 긴 테이퍼 및 고급 팁과 micropipettes를 준비하는 바늘 풀러를 사용합니다. </li><li> 호감 팁 ~ 2mm팁에서 집게를 사용하여 톱니 휴식을 만들 수 있습니다. </li></ol> 미디어 <ol><li> 부화 미디어 : 1X 주식 신선한 4분의 3 일반 수륙 양용 미디어 (NAM)를 준비합니다. 0.025 MG / ML gentamycin을 추가합니다. </li><li> Electroporation 미디어 : 위에서와 0.1 % Benzocaine (시그마, 06950). </li></ol> 2. Electroporation Micropipette 설정 <ol><li> ~ 1 μl 주입 솔루션 micropipette를 입력합니다. </li><li> micromanipulator에 micropipette를 확보하고 microinjector (Picospritzer II)에 첨부해 주시기 바랍니다. </li><li> 탁상에서 50 °의 각도 micropipette. </li><li> 20 PSI로 분사 압력을 설정합니다. </li><li> 펄스 30 NL를 삽입하는 micropipette를 보정합니다. </li></ol> 올챙이 준비 <ol><li> 5 분 electroporation 매체의 잠복기에 의해 무대 28 Xenopus의 애벌레를 마취. </li><li> electroporation 미디어 가득 electroporation 챔버에 anesthetized 올챙이를 전송합니다. 위치 배아긴 시간 잘 수 있도록 머리를 아래 지느러미 측면과 노출된 복부 측면과 함께 T - 교차점에 달려 것을. 머리는 약간 꼬리에 비해 상승한다. </li><li> 집게를 사용하면, 부드럽게 플라스티신 주변과 잘에서 올챙이을 확보. (참고 :.. 올챙이가 보안되지 않은 경우, 그것이 떨리는와 electroporation 동안 전극 연락을 얼굴 조직이 심각하게 손상될 수 있으므로이 경우에는 올챙이를 삭제 수 있습니다) </li></ol> Electroporation <ol><li> micropipette 팁 즉시 시멘트 선에 후부와 얼굴 mesenchyme에 넣습니다. </li><li> mesenchyme로 30 NL 솔루션을 주사. </li><li> micropipette를 철회. </li><li> 빠르게 배아의 머리 (그림 3) 병렬 전극 팁에 맞춥니다. </li><li> 8 50 MS, 20mV 사각형 펄스를 적용합니다. </li><li> 전극을 철회. </li><li> 집게를 이용하는 것은 신중하게 우물에서 올챙이 릴리스 3 / 4 NAM, 0.025 MG / ML gentamycin을 전송. </li><li> Tadpoles는 NAM, 0.025 MG / ML overn 4분의 3에 incubated 수 있습니다항공, 이상. </li><li> 24 시간 후에 형광 현미경에 의한 효율적인 electroporation을위한 화면 배아. </li></ol> 3. 대표 결과 : 형광 분자의 사용은 electroporated 배아 쉽게 검사 수 있습니다. 그림 4는 14.5에서 incubated MO electroporated tadpoles ~ 12, 48 electroporation 후 96시간의 전형적인 배치, ° C.를 보여줍니다 형광 현미경을 사용하여 수법의 살인인 즉시 electroporation 후 시각과 electroporation 후 며칠 동안 계속하실 수 있습니다. 우리의 경험에서 형광 무대 46 (~ 5 일 후)에 약하게 분명하다. 연골에는 형광은 차별화 (~ 스트리트 42)의 발병 이후 크게 감소하지만, MO 형광는 pharyngeal endoderm 등 다른 세포 유형에 더 강력하게 지속. 형광 현미경은 oligonucleotides는 연골 등 여러 craniofacial 조직에 통합됩니다 것을 보여줍니다. Oligonucleotide 형광 C은 종종 머리 양쪽에 조직에서 시각 수 있습니다. 이것은 가능성이 electroporation하기 전에 느슨한 craniofacial mesenchyme 내내 사출 솔루션의 빠른 확산에 의한 것입니다. <img alt="그림 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> 1 집에서 만든 전극을 그림. L - 모양의 텅스텐 와이어는 무독성 점토 또는 퍼티를 사용하여 1 ML의 주사기에 첨부되어 있습니다. (A) 전극 말단은 5mm를 측정합니다. (B) 테르 병렬 실행 등, 전극의 쌍을 연결합니다. 전극 DC 케이블에 의해 펄스 발생기에 연결된 있습니다. <img alt="그림 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> 이 Electroporation 챔버 그림. (A) 플라스티신 줄지어 90mm 요리는 미디어와 시계 제조인 없음 5의 집게와 함께 새겨진 T - 모양의 챔버로 가득합니다. (B) 긴 쪽을 2mm X 2mm에게 X10 mm를 측정짧은 조치 2mm X 2mm X 5mm를 하다니. 배아의 머리가 T - 접합, 복부 측면에 달려있다. <img alt="그림 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> 3 도식 보여주 electroporation 절차를 그림. 성 28 올챙이는 복부 측면은 최고, electroporation 챔버에 배치됩니다. Micropipette는 얼굴 mesenchyme 기본 시멘트 선에 삽입됩니다. 주입. Micropipette은 제거하고 L - 모양의 전극은 병렬로 머리를 측면 정렬됩니다. 팔 50 MS, 20 MV 사각형 펄스를 적용합니다. 전극을 철회. 원하는 단계로 tadpoles 성장. 수법의 살인인 또는 형광 현미경을 사용하여 GFP 발현을 Visualise. <img alt="그림 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> 사 대표 tadpoles 12 (A), 48 (B) 및 96 (C) 시간 게시 electroporation을 (단계 30, 34 각각 44) 그림. ( &quot;- B&quot;) 형광등 MO는 사슴에 craniofacial mesenchyme 내에서 시각 수 있습니다초밖에 30 34. 형광은 스테이지 44 (화살촉, C&#39; - C &quot;)에 연골에서 발견하실 수 있습니다. 창자는 매우 autofluorescent입니다.

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저자는 이익의 갈등이 없습니다.

이 비디오에서는, 우리는 Xenopus tadpoles의 얼굴 mesenchyme에 electroporation - 중재 유전자 전달의 가능성을 증명하고있다. 이 방법을 사용하여, 우리는 나중에 시간 지점에서 특정 조직을 대상 있도록 유전자 기능을 조작의 조기 개발 효과를 무시할 수 있습니다. 우리의 연구는 우리 electroporated 세포뿐만 아니라 관심 단백질에 대한 세포 자율적인 요구 사항의 혈통을 조사 수 있도록, craniofacial mesenchymal 세포의 불일치 인구가 영향을받을 수있는 것으로 나타났습니다. 라이브 영상과 결합, 우리는 craniofacial 개발하는 동안, 시간이 지남에 따라, 유전자 기능을 연구하기 위해이 방법을 사용할 수 있습니다. 이 소설의 방법은 organogenesis의 연구 Xenopus의 tractability를 강조 표시합니다. 우리는이 방법이 널리뿐만 아니라 morphogenesis 및 기타 조직의 분화 연구를 적용할 수 있습니다 것으로 예상.

A correction was made to <a href="http://http://www.jove.com/video/3381">Electroporation of Craniofacial Mesenchyme</a>. There was an incorrect unit used for the voltage.

Erratum: Electroporation of Craniofacial Mesenchyme

electroporation of craniofacial mesenchyme

Electroporation은 정확한 시간 포인트에서 조직에 정확한 위치에 DNA 및 기타 유료 macromolecules을 전달하는 효율적인 방법입니다. 예를 들어, electroporation는 Xenopus의 신경 및 망막 개발 연구에 큰 성공과 함께 사용되었으며, 닭 및 마우스 1-10. 그러나 이러한 연구 모두, 조사관은 부드러운 조직을 타겟팅되지 않은 점에 유의하는 것이 중요합니다. 우리가 craniofacial 개발에 관심이 있기 때문에, 우리는 얼굴 mesenchyme를 대상으로하는 방법을 적응. 우리가 문헌을 검색하면, 우리는 놀랍게도, 연골 조직으로 성공적으로 유전자 전달의 거의 보고서를 발견했습니다. 이러한 연구의 대부분은 chondrogenic 셀 라인으로 siRNA 또는 단백질 전달, 또는 관절염 11-13의 동물 모델로 유전자 치료 연구했다. 같은 닭고기 드나 마우스와 같은 다른 시스템에서는 얼굴 mesenchyme의 electroporation 도전했습니다 (개인 communicat을이온, Craniofacial 개발 부서, KCL). 우리는 Xenopus의 procartilaginous 및 연골 조직에 electroporation 더 효과가있을 것을 가상. 우리의 연구에, 우리는 그 얼굴 연골에 유전자 전달의 얼굴 primordium가 여전히 이전의 연골 분화에 부드러운 조직으로 구성되어 있습니다 초기 단계 (28)에서 효율적으로 발생 보여줍니다. Xenopus은 craniofacial 개발 분석을 위해 매우 접근 척추 시스템입니다. Xenopus의 배아는 초기 단계의 분석을 수 있도록 외부 수정된, 그리고 라이브 단세포 해상도 이미징뿐만 아니라 어머니 14 재사용을 촉진하기 때문에 Craniofacial 구조는 더 쉽게 볼 수 Xenopus의 다른 척추 모델에 비해 주로하고 있습니다. Xenopus의 유충이 디로 크고 쉽게 외부로 개발 척추 모델 중에서 Xenopus은 zebrafish보다 craniofacial 분석에 더 유용합니다ssect 및 개발 얼굴 지방은 고기의 상응하는 영역보다 이미지에 더 액세스할 수 있습니다. 또한, Xenopus은 zebrafish (~ 100,000,000년 가까이) 15보다 인간 evolutionarily 가까이 있습니다. 마지막으로,이 단계에서 Xenopus의 tadpoles는 투명하고, 형광 단백질 또는 분자의 동시 표현이 개발 연골 쉽게 시각화를 허용합니다. 우리는이 접근법 우리가 신속하게 효율적으로 생체내 모델 시스템에서 후보 분자를 테스트할 수 것으로 기대하고 있습니다.

Watch this Scientific Journal Video about Electroporation of Craniofacial Mesenchyme at JoVE.com

Electroporation of Craniofacial Mesenchyme

Craniofacial 연골은 다른 조직들과 긴밀한 접촉을 개발하고 살아있는 동물의 조작 어렵습니다. 우리는 초기 배아 효과를 우회하면서 craniofacial 골격의 성장하는 동안 분자 도구를 제공하는 electroporation을 사용하고 있습니다. 이러한 접근 방식은 우리가 효율적으로 후보 분자를 테스트하실 수 있습니다<em&gt; 생체내 인치</em

Craniofacial Mesenchyme의 Electroporation

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

electroporation-of-craniofacial-mesenchyme

Research

JoVE Journal

Biology

11.8K 조회수.  King's College London. Craniofacial 연골은 다른 조직들과 긴밀한 접촉을 개발하고 살아있는 동물의 조작 어렵습니다. 우리는 초기 배아 효과를 우회하면서 craniofacial 골격의 성장하는 동안 분자 도구를 제공하는 electroporation을 사용하고 있습니다. 이러한 접근 방식은 우리가 효율적으로 후보 분자를 테스트하실 수 있습니다 생체내 인치

비디오: Electroporation of Craniofacial Mesenchyme

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mycobacteria의 Electroporation

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Craniofacial 개발 과정 Ectodermal 상피 조직의 신호 특성을 평가

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

배아 칙의 망막에서 비켜 Electroporation에

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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 ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

게놈 전체 유전자 삭제할에<em&gt; 연쇄상 구균 sanguinis</em&gt; 높은 처리량 PCR에 의한

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

마우스 배아 얼굴 외배엽과 Mesenchyme의 분리

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

sox10의 두개 안면 개발의 시각화 : 단풍 나무 형질 전환 제브라 피쉬의 선은 시간 경과 공 초점 현미경을 사용하여

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D 공 초점 현미경을 사용하여 제브라 피쉬의 두개 안면 형태 형성 분석

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

인간 다 능성에 대한 대안 문화는 전지 생산, 유지 보수 및 유전 분석을 줄기

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

메추라기 오리 키메라를 사용하여 두개 안면 개발에 종 - 특정 포스트 평가

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...