제시된 작품은 건강 연구의 캐나다 연구소에서 자금에 의해 부분에서 지원 / RD 캐나다 파트너십 프로그램의 허약 한 X 연구 재단, RD, 대학 에를 랑겐 - 뉘른베르크의 Interdisziplinäres 젠트 룸에 대 한 klinische Forschung에 제롬 레조 이네 재단은 Rd 및합니다 독일 연구 협회에서 RD와 RE합니다. 저자는 pCMV5-FLAG 벡터 사용할 수 있도록 기술 세포 배양 유지 보수에 대한 지원뿐만 아니라 교수 M. 웨 그너에 대한 잉그리드 Zenger을 감사드립니다. 저자는 또한 특히 비디오 촬영에 도움이되는 지원을 위해 나디아 슈뢰더을 감사드립니다.

1. 재고 솔루션 <ol><li> 2 μg의 / μL의 최종 농도 멸균 H의 동결 건조 활성 모티브 분말 2 O를 재현 탁. 혼합주의 깊게 누릅니다. </li><li> (예를 들어, 12 ㎕를 각각 2 μL가 반응에 따라 필요) 작은 분취 량을 준비하고 -20 ° C에 저장합니다. </li></ol> 세포의 2. 준비 <ol><li> 항생제를 포함하여 24 웰 플레이트에 500 μL의 성장 매체에 HEK293 세포 또는 차 신경 세포로 씨 포유 동물 세포. 주 : 신경 세포를 사용하는 경우, 낮은 밀도의 세포를 시드 및 24 웰 플레이트의 두 개의 중간 열을 사용한다. 각각의 잘 따라서 배양 (단계 4.2) 동안 매체를 항구 빈 대응을해야합니다. 신경 세포를 취급 할 때는 항상 세포가 몇 분 이상 인큐베이터 밖에 유지되지되어 있는지 확인하십시오. </li><li> 세포 앱까지 적절한 배양 조건의 세포 (가습 된 5 % CO 2, 37 ℃). 50 %지류. 주 : 신경 세포를 사용하는 경우, 문화 원하는 발달 단계까지 세포에 도달하고 응용 프로그램을 변경할 수 있습니다. 두 매질의 25~50% 주. 매체 (1X B27, 5 mM의 L- 글루타민 및 1X 페니실린 및 스트렙토 마이신을 함유하는 (REF) (16)과 비교)로 Neurobasal 배지. </li></ol> 3. 전차 단지 형성 <ol><li> 반응 당 PBS 50 μL에 각각 POI 또는 해당 항체뿐만 아니라 제어 단백질, 또는 대조군 항체의 0.1 μg의 희석. 참고 :이 기술은 또한 거대 분자 복합체 사전 구속 일차 및 이차 항체 (. CP &#39;대표 결과&#39;그림 1)으로 구성된 작동합니다. 믹스와 스핀. </li><li> 각 샘플에 대해, 전차의 자동 연관을 방지하기 위해 별도의 튜브를 사용하여 PBS 50 μL 2 μL 전차 원액을 희석. 믹스와 스핀. </li><li> 피펫에 의해 전차의 희석에 희석 POI 또는 항체 (3.1 단계)를 전송팅. 믹스와 스핀. </li><li> 실온에서 30 분 동안 인큐베이션 혼합물 (POI 전차 - 복합체 형성 할 것이다). </li></ol> 셀의 4. 형질 <ol><li> 전차 단지 사전 예열 성장 매체 100 μL (37 ° C, 무첨가)에서 (단계 3.4) 희석. 매체를 제거하고 한 번에 미리 예열 1X PBS를 사용하여 세포를 씻는다. 주 : 신경 세포를 사용하는 경우, 배지를 폐기하지 않는 것이 재사용 될 것이다. 37 ℃에서 보관하십시오. 세척, PBS가 0.5 밀리미터의 MgCl 2, 염화칼슘 (2)를 포함하는 사용합니다. 제안 : 접시 (4.5 단계)을 배양하는 동안 빈 우물에 매체를 보관합니다. </li><li> 세포에 전차 착체 용액 (단계 4.1)를 적용하고, 용액의 균일 한 분포를 보장하기 위해 조심스럽게 흔들어 세포. 1 시간 동안 표준 조건 (단계 2.2)에서 세포를 성장. 10 %의 최종 농도로 혈청을 추가한다. 신경 세포를 사용하는 경우, 단계 4.1에서 매체를 추가 할 수 있습니다. 1 ~ 2 시간 이상 세포 성장. 참고 :최적의 배양 시간은 규모와화물의 특성에 따라 실험적으로 조정할 필요가있다. 다음의 제안은 가이드 라인으로 역할을 할 수있다 : 펩타이드를 들어, 1 시간 ~ 2 시간이 항체, 2 시간을 권장하고, 항체와 신경 세포의 형질 4 시간 동안하는 단백질, 일반적으로 충분합니다. </li><li> 평소와 같이 분석을위한 프로세스 세포. 참고 : 기술은 고정 프로토콜뿐만 아니라 라이브 영상을 사용하여 실험과 호환됩니다. </li></ol> 5. 이미지 <ol><li> 레이저 주사 현미경을 사용하여, 약 0.25-0.5 μm의 거리를 사용하여 세포 및 / 또는 관심의 세포 구획 Z-스택을. 정확한 층 거리 재구성 될 구조물의 크기에 따라 개별적으로 결정되어야한다. </li></ol> 6. 재건 <ol><li> 다음 단계에서 O를 클릭하여 여러 개체를 선택하려면 선택과 이동, Cntr을 사이를 전환 할 Esc를 바닥을 사용n은 그들과 Shift 키 객체 (CP를. 6.10 단계) 잘라. </li><li> Imaris에서 LSM-파일을 엽니 다. </li><li> 밝은 구조가 포화에 도달 할 때까지 각 채널의 표시를 조정하여, 화상을 대조. 표면 구조에 영향을주지 않습니다이 조정은, 그것은 단지 이미지를 임계 화에 실험을 지원하는 역할을 양해 바랍니다. </li><li> 새로운 표면 추가 아이콘을 클릭합니다. 마법사가 열립니다. </li><li> 선택 : 관심의 세그먼트 만 지역. 다음 단계로 진행합니다. </li><li> 관심의 목적에 맞게 선택 상자의 여백을 조정합니다. 이미지는 3 차원이 숙지하고 선택뿐만 아니라 Z 평면에서 조절해야한다는 부담주십시오. 선택 박스의 직사각형이 제대로 관심의 대상이 일치하지 않아야하는 경우, 객체를 회전 및 / 또는 모든 필요한 구조를 포함 할 때까지 선택을 확대. 원치 않는 개체가 나중에 제거 할 수있다 (CP. 6.10 단계). 발하다. </li><li> 해당 채널을 선택합니다annel. 표면적의 상세 수준 또는 구 직경은 구조가 재구성되고 일치하도록 설정해야합니다. 상기 신호 특성에 따라 절대 강도 또는 로컬 콘트라스트 중 하나가 사용될 수있다. 일반적으로, 지역 대비 확산 신호에 대한 더 나은 작동합니다. 어떠한 경우에도, 설정이 각각 관심있는 각각의 신호 또는 구조에 대해 개별적으로 조절 될 필요가있다. </li><li> 임계 화 도구를 사용하여 관심의 구조를 재구성. </li><li> 녹색 화살표 아래를 클릭하여 복원을 완료합니다. </li><li> 절단 및 필요에 따라 표면을 제거하기 위해 연필 도구를 사용하여 추가 개체를 조정합니다. 원하는 개체를 절단하기 위해 이미지를 따라서 남북 방향으로 절단 기울에만 가능합니다. </li><li> 선택 통계, 자세한 통계 및 평균 값에 의해 각 표면 볼륨 영역의 측정과 강도를 얻습니다. </li><li> 색상 코드 통계를 관찰하려면 (선택 단계) (CP. 그림 2E </strong&gt; 및 F)는, 해당 표면의 색상 탭을 선택하고 대신 &#39;자료&#39;의 &#39;통계 코딩&#39;을 표시합니다. 몇 가지 옵션이 표시됩니다. </li></ol>

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The authors have nothing to disclose.

여기서는 도즈 구동 방식으로 세포 기능을 조절 단백질 발현 수준의 중요성을 연구 프로토콜을 제시한다. 기술 된 프로토콜은 그러므로 세포 수준에서 단백질 기능의 상세한 연구를 촉진 해마 신경 세포 등 다양한 종류의 포유류 세포에서 단백질 발현의 미세 조정 조작을 허용한다. RNA 간섭의 POI를 하향 조절하는 공지의 방법을 나타낸다 있지만 단점 (CP있다. 14)를 ?...

모든 살아있는 유기체 자체 개발 명령뿐만 아니라 환경 신호에 반응하는 단백질 발현의 엄격한 제어가 필요하다. 따라서, 메커니즘은 다수 정확하게 앱 부호화 각 단백질의 발현을 조절하는 진화 중에 고안되었다. 그 삶의 주어진 시간에 모든 진핵 세포에 존재하는 20,000 유전자. 단백질 생산의 다른 단계에서 발생하는 규제 메커니즘은 번역 후 단백질 수정, 전송 및 분해의 방향으로 처리 크로 마틴 구조, 전사 및 RNA의 관리에 이르기까지 다양합니다. 분자 기계 및 변형 단백질 발현 수준을 기본에 고장이 암 또는 지적 장애 등 다양한 질병과 연관되어 있는지, 따라서 놀라운 일이 아니다. 사실, 신경 세포의 개발 및 포유 동물의 뇌 기능의 뛰어난 복잡성에서 찾고 SENSI단백질 발현의 변화에​​ 이러한 정교한 시스템의 tivity는 알츠하이머와 허약 한 X 증후군 (FXS) 같은 파킨슨 병 (AD 및 PD)뿐만 아니라 자폐증 스펙트럼 장애 (ASD) 등 여러 가지 잘 알려진 지적 적자에서 드러난다. 후자 질병은 하나의 번역 조절 단백질 FMRP (유약 X 정신 지체 단백질) 1-4의 손실로 인한 ​​단백질의 다양한 광범위한 misexpression 특징으로한다. 점 돌연변이는인지 장애 환자에서 식별되지 않은 상태에서 5 캐핑 mRNA의 수정에 의해 mRNA의 안정성과 변환을 관리 또한, 가변 전하에 영향을 미치는 염색체 재구성 연결된 x를 단백질 A (VCXA) 단백질은 최근 지적 결핍과 연관되고 지금과 같은 6, 7, 관찰 된 정신 장애는 변경된 VCXA 발현과 그 대상 보호 자전거의 조절 이상 발현에서 발생한 것을 건의인. 이러한 연구 결과, 드 노보 ASD와 관련된 유전자의 복제 수 변화에 따라서 상승 또는 감소 단백질 발현 수준이 발생할 수 있음을 아이디어를 지원하는 새로운 유전자 중복 및 삭제 ASD (8)의 중요한 위험 인자임을 설립 여부를 조사 연구와 일치 지적 적자. 놀랍게도, 최근의 연구는 또한 특정 단백질의 발현 량을 정확하게 거의 안전 여유도 9 높은 단백질 량의 결과로서 그 응집을 방지하도록 조절되어 있다는 증거를 제공 하였다. 따라서, 심지어 작은 단위는 AD 및 PD (9)과 같은 질병을 유발하기에 충분하다는 것을 제안되었다. 단백질 발현 조절에 기여하는 분자 기계의 다양한 이러한 결과에 비추어 복잡한 조절 방식을 제안했지만, 5000 포유류 유전자 (10)의 발현 량을 조사하는 연구는 것을 증명자연은 더 인색 방식을 선호 : 단백질의 세포 풍요 로움을 주로하여 RNA 가용성 관리는 주로 미세 조정하는 단백질 발현을 제공한다는 설명, 번역 (10)의 수준에서 조절되는 것으로 나타났다. 관심 단백질의 용량을 연구 장소 (POI)를 따라서 만 내인성 단백질의 기능을 이해하는 것이 중요하지만, 또한 많은 질병의 연구 및 치료의 개발이 아니다. 따라서, 지난 수십 년은 POI의 복용량을 조작하는 RNA 간섭을 사용하여 몇 가지 전략의 발전을 보았다. RNA 간섭 널리 단백질의 기능을 연구하기 위해 사용되며, 심지어 암, 안과 질환의 치료뿐만 아니라 환자의 11-13 항 바이러스 요법을 추구하는 임상 시험에 적용되고 있지만, 약간의 어려움이 전략 불가능 렌더링 할 수있는 발생할 수있다. 예를 들어, 상 동성으로 최저 구동 시드 시퀀스는 compara은상상력 짧은, 따라서 홍보 오프 대상 효과. 고효율 서열이 드문 및 시간과 비용이 많이들 수있는 권리 순서를 식별 (14 리뷰) 옵션의 수천 사이에서 발견해야하지만 이후 결과는 여전히 실망 할 수있다. 또 다른 전략은 직접 항체에 의해 POI를 대상으로하는 것입니다. 여기서, 우리는 세포 단백질의 가용성을 감소 (활성 모티프 제조) 단백질 담체 전차의 사용을 설명하고, 삼차원 재구성의 고용 녹다운 따라 단백질의 기능을 연구. 전차, 자체 2.8 kDa의 펩타이드의 활성 모티브, 포유 동물 세포 (15)의 세포막을 가로 질러 셔틀 펩티드, 단백질 및 항체로 사용된다. 전차-POI 단지 가는데, 소수성 상호 작용을 이용하여 비공유 결합 된 거대 분자 복합체를 형성함으로써 관심 장소와 펩타이드 관계자는 엔도 좀-IND 세포에 내장되어 있습니다ependent 방법. 중요한 것은, 전차가 왕복 단백질의 세포 내 위치 파악에 영향을 미치지 않으며, 세포 독성 효과를 발휘하는 또는화물 (15)의 생물학적 활성에 영향을 미치는 것으로도 나타내었다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

단백질 발현의 엄격한 관리가 살아있는 유기체의 모든 필수적인 아니라 세포 모델에서 단백질의 기능을 조사하는 중요한 전략뿐만 아니라. 따라서, 최근의 연구는 RNA 및 항체 간섭을 포함하여 포유 동물 세포주 또는 동물 모델에서의 단백질 발현을 대상으로 다른 도구를 발명했다. 첫 번째 전략은 지난 20 년 동안 많은 관심을 수집하고 있지만, 세포막에서 세포에 항체화물의 전위를 매개 펩타이드는, 훨씬 적은 관심을 얻을. 이 공보에서는 항체 간섭 실험을 수행하기 위해, 인간 배아 신장 세포에서뿐만 아니라 차 해마 뉴런 전차라는 펩티드 담체를 이용하고 상기 단백질의 기능을 분석하는 삼차원 재구성의 적용을 설명하는 방법에 대한 상세한 프로토콜을 제공한다. 우리의 연구 결과는 전차는 아마도 핵 지역화 신​​호, particula에 있음을 시사RLY 소마와 핵에 존재하는 단백질을 대상으로 매우 적합합니다. 차 해마 문화에 전차를 적용 할 때 놀랍게도, 시약은 놀라 울 정도로 잘 해리 신경 세포에 의해 허용 될 밝혀졌다.

다음 단락에서, POI의 기능 최저 설명 예시 결과 전차 시약 및 항체의 간섭이되게됩니다 사용 (Simiate는, 자세한 내용은 16, 17 참조). 연구 결과는 Simiate의 감소 발현이 전사 활성을 저해하고, 투여 량 의존적으로 높은 항체 양 (&gt; 1 μg의 항체)을 적용 할 경우 99 %의 사망률에서 남중, 세포 사멸을 유도 입증 (이 발견은 이전에 발표되었다 (16)). 여기서는 상세하게 사용?...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

공부 단백질 기능 및 항체의 간섭과 3 차원 재건에 의해 변경 단백질 발현의 역할

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

studying-protein-function-role-altered-protein-expression-antibody

Research

JoVE Journal

Bioengineering

6.6K 조회수.  University of Erlangen-Nuremberg. Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

비디오: Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

막 단백질 폴딩의 열역학은 형광 분광학에 의해 측정

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

연구

생체공학

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

난소와 Oviducts의 3 차원 조직 문화 알긴산의 Hydrogels

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

마이크로 유체 플랫폼을 사용하여 높은 처리량 단백질 표현 생성기

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

단백질 접기 유학을위한 Microfluidic 믹서

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

모델 보컬 폴드 폴립에 의해 유도된 3차원 유동 분리 조사

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

사례 연구로 담배에 과도 단백질 발현 : 실험 접근의 디자인을 사용하여 복잡한 시스템의 특성

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.