Name: transient expression of proteins by hydrodynamic gene delivery in mice
Uploaded: 2014-05-05T16:30:00
Description: Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

우리는 처음에 우리에게 기술의 다양한 측면에서 자신의 지속적인 안내 HGD 기술과 러셀 박사 톰슨 (의학의 알버트 아인슈타인 대학)을 가르치는 시아 리우 (Regeneron 제약) 감사합니다. 이 작품은 헌터 칼리지에 의해 투자되었다, CUNY는 기금을 시작하고 NSF 빵 상 IOS-1249166. 우리는 마우스의 간에서 조직학에 대한 NYULMC 조직 병리학 코어 NYUCI 센터 지원 그랜트, NIH / NCI 5 P30CA16087-31 감사합니다.

여기에 설명 된 모든 실험은 헌터 칼리지의 기관 동물 케어 및 사용위원회, 뉴욕 시립 대학에 의해 승인되었습니다. 독소가없는 플라스미드 DNA의 1. 준비 <ol><li> 갓 줄무늬 선택적 판에서 포유 동물 발현 벡터에 대한 관심의 유전자를 포함하는 박테리아의 하나의 식민지를 선택합니다. </li><li> 박테리아의 성장과 수확에 대한 시판 독소가없는 플라스미드 정제 키트의 수첩에...

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B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14, 129-137 (2007).</li></ol>

올바르게 수행 할 경우, HGD는 유전자 전달의 매우 안전하고 효과적인 방법입니다. 성공적인 HGD에 중요한 단계는 1)보다 8 초에 마우스 3)의 꼬리 정맥에) 생리 식염수 차량 2의 큰 볼륨에서 DNA의 정확한 양을 제공. 주입 과정 자체가 대단한 약간의 손재주가 필요하지만,이 여섯 번째 절차의 성공은 종종 실험주의 준비에있다. 최대한의 유전자 발현을 달성하기 위해 필요한의 pDN...

에 의해 처음으로 설명하기 때문에 류 등. 및 장 등., 유체 유전자 전달 (HGD)는 설치류 모델 시스템 1, 2 유전자의 기능을 연구하는데 매우 중요한 도구가되고있다. 기술은 표적 기관 1, 2의 세포에 의해 플라스미드 DNA의 흡수를 촉진하기 위하여 마우스의 꼬리 정맥에 용액의 대량 (8~12% 체중)의 급속 주입 (5-7 초)을 포함한다. 이러한 조건은 신장, 비장, 폐와 심장에 간 적은 유전자 발현의 강력한 유전자 발현을 초래할. 10 μg을 pCMV-LacZ를 플라스미드 주입 HGD 날짜 1에 가장 효율적인 비 바이러스 성, 생체 내 유전자 전달 방법을 만드는 간세포의 많은 40 %가 감염 될 수 있습니다. 바이러스 성 항공사와는 달리, pDNA를 준비하기 쉽고, 설치류 호스트 3 면역 반응을 유도하지 않고 끝이 재결합하여 건강 위험하지 않습니다ogenous 바이러스. HGD에 의해 전달 된 DNA 분자가 포장을 필요로하지 않기 때문에 또한,이 방법은 150킬로바이트 4만큼 많은 세균 인공 염색체 (BAC)의 전달에 적합하다. 유체 역학적 방법에 의해 전달 된 분자의 다른 종류의 RNA 5-10, morpholinos와 11, 단백질 12, 13 및 다른 작은 분자 (12, 14)이 (가) 있습니다. 다른 전달 방법에 HGD의 장점과 단점은 문학 15 ~ 20 우수 리뷰에서 논의되었고, 저자의 수는 21-23 절차에 대한 자세한 설명을 제공하고 있습니다. HGD에 의해 마우스에 형질 전환 유전자를 도입하면 안전하고 1-3, 24 효과적이 방법은 비교 성공 (25)와 쥐에 사용되어왔다. 특정의 수정, 개념 증명 실험은 닭 26, RAB에서 실시 된큰 동물이 기술의 생체 내 응용 프로그램이 과제로 남아,하지만, (27)와 돼지 28 비트. 이 방법을 사용할 때, 또 다른 일반적인 제한은 가능한 포유류 발현 벡터의 많은 유전자 발현의 지속성, 높은 수준을 달성하기 성분이 부족하다는 것이다. 대상 기관에서을 pCMV 룩 플라스미드 유전자 발현을 사용하지만, 초기의 높은 발현 수준이 주입 한 후 첫 주에 급격히 인하, HGD 후 십분 빠르면 분명하다. 장기간의 형질 전환 유전자의 발현은 플라스미드 디자인 3, 24 그러나, 높은 수준의 유전자 발현의 유지가 종종 필요 반복 주사에 사용되는 프로모터와 인트론에 따라 가능하다. 이러한 이유로, HGD가 손상 단백질이나 단백질 제품에 장기간 노출의 결과 만성 질환을 연구 덜 적합 할 수 있습니다. 이러한 한계로, HGD는 PO 공부를위한 매우 강력한 도구입니다유전자 및 그것의 효과 tential 역할 (검토를 위해, 15 참조) 생체 내뿐만 아니라 치료 효과와 단백질의 조절에 질병의 동물 모델을 확립하는 돌연변이. 예를 들어, HGD 개별적으로 각각의 유전자가 녹아웃 한 마우스에 다양한 유전자 구성체를 도입하여 도메인 및 단백질의 아미노산에 기능을 할당하는 데 사용될 수있다. 또한,이 기법은 임의의 마우스 균주에서 사용될 수있다. 배달의 정맥 주입 볼륨과 속도에 정확한 바늘 삽입 :이 프로토콜은 성공적으로 형질 전환을 달성하기 위해 필요한 기술적 인 측면에 초점을 생쥐에 HGD에 대해 설명합니다. 이 방법의 적용은 아프리카 트리파노소마, 인간의 치명적인 질병과 가축 (29, 30)의 마우스 모델에서 입증된다. trypanosomes의 몇몇 종 가축에 질병을 일으키는 원인이 있지만, 대부분은 B의 면역 복합체를 타고난에 의한 인간의 질병을 일으킬 수CTRL 키를 누른라고 trypanosome 세포 용해 인자 (TLFs) 29, 31, 32. 이러한 기공 형성, 고밀도 지단백질 (HDL)의 두 가지 고유, 영장류 특정 단백질이 들어 있습니다. trypanosomes에 TLFs의 흡수를 용이하게 HPR, 리간드, 및 APOL-I, 용해성 구성 요소 31, 33 ~ 38을 Trypanosoma을 brucei의 rhodesiense 인해 바인딩과 인간 APOL-I (34), (39)을 중화 혈청 내성 관련 단백질 (SRA)의 표현으로 인간을 감염시킬 수 있습니다. 비비 TLF는 인해 발산 APOL-I 단백질 40 SRA에 의해 중화되지 않습니다. 포유 동물 발현 벡터 (pRG977), 쥐 원숭이 TLF 구성 요소의 형질 전환 식을 사용하여, 이전에보고 된 인간 감염 trypanosomes (40)에 대한 보호 기능을 부여한다. 여기에 제시된 대표적인 데이터는 유체 역학적 유전자 전달이 단백질의 치료 효과를 연구에 적용 할 수있는 방법을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcohol Prep Wipe</td>
			<td style="width:113px;">Webcol</td>
			<td align="right" style="width:119px;">6818</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AST kit, Amplite Colorimetric</td>
			<td style="width:113px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">13801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical Tube, 50 ml</td>
			<td style="width:113px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:113px;">Qiagen</td>
			<td align="right" style="width:119px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:113px;">KIMTECH</td>
			<td align="right" style="width:119px;">34120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse Tail Illuminator</td>
			<td style="width:113px;">Braintree Scientific</td>
			<td style="width:119px;">MTI RST</td>
			<td style="width:167px;">Or equivalent</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 20 Gx 1 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 27 Gx 1/2 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">pRG977 (mammalian expression vector)</td>
			<td style="width:113px;">Regeneron Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Material Transfer Agreement required</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium Chloride, 0.9% INJ USP 10 ml, Hospira&#160;</td>
			<td style="width:113px;">Fisher</td>
			<td style="width:119px;">NC9054335</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Syringe, 3ml, Luer-Lok Tip</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">309657</td>
			<td style="width:167px;">Luer Lock necessary to withstand pressure from HGD</td>
		</tr>
	</tbody>
</table>

transient expression of proteins by hydrodynamic gene delivery in mice

생체 내에서 형질 전환 유전자를 효율적으로 발현은 유전자 기능을 연구하고 질병에 대한 치료법 개발에 매우 중요합니다. 지난 몇 년 동안, 유체 유전자 전달 (HGD)이 설치류에 형질 전환 유전자를 전달하기위한, 간단하고, 빠르고, 안전하고 효과적인 방법으로 떠오르고있다. 이 기술은 관류 기관의 세포막의 투과성을 증가시키는 생리 용액의 대량의 급속한 주입에 의해 생성되는 힘에 의존하고, 따라서 세포로 DNA를 전달한다. HGD의 주요 장점 중 하나는 알몸 플라스미드 DNA (pDNA를)를 사용하여 포유류 세포로 형질 전환 유전자를 도입 할 수있는 기능입니다. 플라스미드를 사용하여 외래 유전자를 도입하는 방법은 매우 안전하고, 최소한의 고효율, 힘들고, 바이러스 사업자에 반대합니다. HGD가 처음 생쥐에 유전자를 전달하기 위해 사용 된, 그것은 이제 올리고 뉴클레오티드, 인공 염색체, RNA, 단백질 및 마우스에 작은 분자를 포함한 물질의 넓은 범위, 래트를 전달하는 데 사용와, 제한된 정도, 다른 동물. 이 프로토콜은 생쥐의 HGD에 대해 설명하고 프로 시저를 성공적으로 수행하는 중요한 방법의 세 가지 주요 측면에 초점을 맞추고 : 정맥에 바늘의 정확한 삽입, 분사의 볼륨 및 배달의 속도. 예를 분비, 영장류 특정 단백질을 인코딩하는 두 유전자의 일시적 발현이 방법의 응용 프로그램을 보여 주어, 아포 지단백 LI (APOL-I)과 합 토글 로빈 관련 단백질 (HPR).

마우스의 꼬리 정맥에 바늘의 정확한 위치는 (도 1에 도시 된 바와 같이)을 성공적으로 유체 역학 기반하여 형질 도입 유전자 전달을위한 필수 조건이다. 주입의 전체 볼륨은 6-8의 기간 내에 전달 될 수 있도록 종종 기술의 가장 어려운 부분은, 그러나, 움직임이없는 꼬리 정맥 내 바늘의 유지이다. 사출 과정에서 사소한 오류가 크게 감소 된 형질 전환 효율 및 단백질 발현의 원인이 ?...

Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

유체 유전자 전달에 의한 벌거 벗은 DNA의 생체 내 형질에서 최소한의 염증 반응과 동물의 조직에 유전자를 소개합니다. 유전자 산물의 충분한 양의 유전자 기능 및 조절뿐만 아니라, 단백질의 구조 및 기능 분석 될 수 있도록 생성된다.

마우스의 수력 학적 유전자 전달에 의한 단백질의 일시적 발현

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

transient-expression-of-proteins-by-hydrodynamic-gene-delivery-in-mice

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

연구

생물학

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

재조합 단백질의 높은 수준의 과도 발현 식물의 효율적인 Agroinfiltration

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

대사율의 측정에<em&gt; 초파리</em&gt; Respirometry를 사용하여

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.