This work was supported by NIH R01-HL119813 (to T.J.B.), American Lung Association RG-196051 (to T.J.B.), a CIMED Pilot and Feasibility grant (to T.J.B.), American Heart Association Predoctoral Fellowships 14PRE19970008 (to Z.Y.) and 15PRE22110004 (to D.L.K.).

대규모 과도 형질에 대한 플라스미드 DNA의 밀리그램 수량 1. 생산 <ol><li> 제한 부위 클로닝, 또는 다른 적절한 기술을 사용하여 높은 카피 수 포유 동물 발현 벡터에 목적 단백질을 복제. <ol><li> 최적의 결과를 가지고 pHLsec 7 벡터를 사용하는 내장 된 C- 말단 6His 태그, 강력한 프로모터 코작 순서 및 최적화 분비 신호. </li></ol></li><li> 적격 세포에 플라스미드를 변형. <ol><li> 플라스미드 DNA의 1 μg의 관할에 대장균의 20 μl를 첨가하고 30 분 동안 얼음 위에서 배양한다. </li><li> 42 ℃에서 열 쇼크 셀 35 초 후, 2 분 동안 얼음 위에서 배양한다. </li><li> 미생물 성장 매체 (SOC)의 300 μl를 추가하고 220 rpm으로 떨고, 45 분 동안 37 ° C에서 품어. </li><li> 적절한 항생제 선택과 한천 플레이트에 플레이트 세포. <ol><li> 플라스미드 pHLsec 벡터의 경우 100 μg의 / mL의 카르 베니 실린을 사용하여. </li></ol></li></ol></li><li> 루리아 국물 (LB) 미디어 250 ml의 문화 식민지는 220 rpm으로 흔들어, 37 ° C에서 100 μg의 / ㎖ 항생제 (카르 베니 실린) O / N로 보충. </li><li> 제조 업체의 프로토콜에 따라 고속 플라스미드 맥시 키트를 사용하여 문화의 DNA를 정화. <ol><li> 대신 버퍼 TE의 버퍼 EB (10 mM 트리스 - CL, 산도 8.5)에서 DNA를 용출. </li><li> -20 ° C에서 형질 전환 및 저장에 필요한 양의 정제 된 플라스미드를 나누어지는. </li></ol></li></ol> 293F 세포의 2. 대규모 문화와 과도 형질 <ol><li> 10 글루타민 ㎖의 5 ml의 펜 / 연쇄상 구균 (둘 다 100 배)와 보충 1 L 293F 미디어. 4 ℃에서 보관하십시오. 5 ㎖ 펜 / 스트렙토 단백질 수율을 향상 무 혈청 조건으로 환원 항생제 농도가 형질 중에 세포 생존을 개선에 충분한 강도이다. </li><li> 1 리터의 폴리 카보네이트 300 ml의 미디어 문화 293F 세포는 배출 캡으로 삼각 플라스크를 당황8 % CO 2와 37 ° C에서의 표준 조직 문화 인큐베이터에 떨고있다. </li><li> 세포에 5 × 105 / ㎖ 밀도 형질 전에 하루 희석. </li><li> 293F 세포 미디어 부스트 V / w 2 %의 10 % 부피를 첨가하여 형질 감염, 보충 배지 날. <ol><li> 500 밀리그램 / dL로의 글루코스 농도를 달성하기 위해 필요에 따라 제조자의 지시 및 사용에 따른 보충 글루코스 모니터를 이용하여 글루코스 농도를 측정한다. </li></ol></li><li> 단백질 당화을 제어하는​​이 단계에서 kifunensine (1 μg의 / ml의 최종 농도)를 추가합니다. </li><li> DNA의 계산 볼륨 1 × 10 6 세포 당 1 μg의 플라스미드 필요합니다. 멸균 조건하에, 5 ㎖의 무 혈청 배지에서 희석 DNA. </li><li> 형질 전환 시약 μL 2 당 1 μg의 플라스미드에 필요한 형질 전환 시약의 양을 계산합니다. 멸균 조건하에, 5 ㎖의 무 혈청 배지에서 형질 전환 시약 (PEI-TMC-25)를 희석한다. </li><li> 더하다1 ml의 단위로 DNA 용액으로 형질 전환 시약, 부드럽게 혼합. 형성 시약-DNA 복합체에 대한 RT에서 30 분 동안 배양한다. 이어서 적가 방식으로 셀에 용액을 추가한다. </li><li> 형질 세포는 72-96 시간 동안 단백질을 발현하도록 허용합니다. ~ 10 % 볼륨 셀 부스트 매일 미디어, 또는 필요에 보충은 혈당이 400 ~ 600 밀리그램 / DL을 읽고 유지합니다. </li></ol> 3. 정제 <ol><li> 세포를 펠릿 화하고 상층 액을 수집 1,300 XG에서 20 분 동안 원심 분리기로 원심 분리 플라스크 배양을 경사 분리한다. 필요하다면, 두 번째 회전 및 / 또는 상등액을 명확히 0.22 μm의 필터를 사용한다. </li><li> 10 %의 10 배 부피의 Ni-NTA 추가 결합 완충액 (1.5 M의 NaCl, 0.5 MK 2 HPO 4, 0.1 M 트리스 pH가 8.5, 50 mM의 이미 다졸). </li><li> 열에서의 Ni-NTA 슬러리 2 ㎖를 추가하고 1X 바인딩 버퍼의 10 열 볼륨 (CV)로 평형화하여 중력 열을 준비합니다. 가능하면, 4 ° C를 방에서 모든 열 단계를 수행. Alternativ엘리, 진정 단백질 및 열 단계 전에 얼음에 모든 버퍼 및 단백질을 유지하고 수집 흐름을 통해 얼음에. 주 : 니켈 NTA 슬러리를 50 체적 %의 수지 및 제조의 명시 결합능은 50 ㎎ / ㎖이다. NI-NTA 비드 여러 용도로 재충전 될 수있다 </li><li> 수지 위에 뜨는 흐름과 흐름을 통해 수집합니다. 이 단계를 반복합니다. </li><li> 세척 버퍼의 10 CV로 세척 (300 mM의 염화나트륨, 50 mM의 K 2 HPO 4, 20 mM의 이미 다졸 산도 8). </li><li> 용출 완충제 5 CV로 단백질을 용리 (300 mM의 염화나트륨, 50 mM의 K 2 HPO 4, 250 mM의 이미 다졸의 pH 8). </li><li> 경우 탈 글리코 실화가 필요합니다 : <ol><li> 0.5 ㎖의 최종 부피를 위해, 원심 분리 농축기를 이용하여 0.43 ml의 용출액을 농축시킨다. 양식을 침전 경우, 16,000 XG, 4 ℃에서 원심 분리하여 이물질을 펠렛. </li><li> 500 밀리미터 나 - 구연산 산도 5.5의 50 μl를 추가합니다. </li><li> EndoHf (1 × 106 U / ㎖)의 20 μl를 추가합니다. 2 시간 동안 실온에서 품어. 아니E : 효소는 농축 된 단백질이 응집시킬 수있는, 37 ℃에서 최적으로 작동합니다. SDS-PAGE 또는 면역 탈 글리코 실화에 의해 평가가 불완전한 경우, RT 배양을 연장한다. 효소는 4 ° C에서 활동이 없습니다. </li><li> 인산염 완충 식염수 (PBS) 또는 최종 저장 버퍼에 세척 아밀로스 수지 배 : EndoHf를 제거하십시오. 4 ℃에서 1 시간 동안 수지와 단백질을 품어. 구슬 펠렛 상층 액을 수집하기 위해 1,000 XG에서 5 분 스핀. </li><li> 적절한 분획 분자량 원심 분리 필터를 이용하여 단백질을 농축시키고, 저장 버퍼 (150 mM의 염화나트륨, 20 mM의 HEPES pH를 7.5)로 교환 버퍼. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

HEK 293F 세포는 번역 후 변형을 필요로하는 단백질의 강력한 생산을 제공한다. 이 시스템은 이황화물, 당화, 그렇지 않으면 더 일상적인 표현 도구를 사용하여 결석 것 인산화를 포함 기본적으로 접힌 단백질의 신속하고 확장 가능한 표현을 할 수 있습니다. 또한,이 시스템은 단순히 여러 플라스미드의 공동 형질 감염에 의해 발현 및 다중 단백질 복합체의 정제를 위해 사용될 수있다. TREM2 외에도,?...

원자 수준에서 단백질의 구조를 이해하는 것은 생물학적 경로와 질병의 분자 기반을 폭로의 핵심입니다. X 선 결정학은 단백질 거대 분자의 구조를 결정하기위한 가장 널리 사용 / 적용 가능한 방법이다. 이 방법의 가장 큰 문제는 제대로 접혀, 순수한 단백질의 충분한 양을 얻을 수있다. 특히 특정 번역 후 수정을 거쳐 분비 포유류 단백질, 작업 문제가된다. 세균 발현 단백질은 단백질 데이터 뱅크 1에서 증착 된 결정화 된 단백질의 주요 원이다. 그들은 저렴하고, 빠르고 일반적으로 단백질의 높은 수율을 생산하기 때문에 세균 발현 시스템은 크게 바람직하다. 그러나, 박테리아에서 발현 포유류 단백질의 세포 외 도메인은 종종 제대로 fo를 균일하게 얻기 위해하는 경우 폴딩 및 광범위한 정화 단계가 필요, 접힌되지 않습니다lded 단백질. 또한, 많은 포유류 단백질은 적절한 폴딩 2를 달성하기 위해 번역 후 당화가 필요합니다. 효모 또는 곤충 세포에서의 발현은 글리코 실화 및 접힘 문제를 극복 할 수 있지만, 당화 포함 번역 후 변형은, 부정확 한 또는 불균일 한 변형을 가진 단백질을 수득 포유류 세포 (3)의 것과 크게 다르지. 포유 동물 세포가 적절한 번역 후 변형 및 폴딩을 위해 필요한 모든 분자 기계를 표현; 그러나, 이러한 발현 시스템은 전형적으로 제한 수율 및 시약 및 소모품의 높은 비용, 대부분의 실험실에서 바람직하지 않다. 폴리에틸렌 이민 (PEI)는 표준 트랜 스펙 션 시약을 비교적 염가이지만 상당한 독성, 낮은 형질 감염 효율, 셀 매체, DNA의 비용 상승을 초래하고 장비의 배양을 부과한다. PEI에 많은 대안이 엄청나게 비싸다. 우리는 해결개선 된 세포 배양 용 도구의 조합을 나타내는 화학적, 단일 단계 정제 한 후, 분비 된 포유 동물 단백질의 발현을위한 신속하고 비교적 저렴​​한 방법에 대한 PEI를 수정함으로써 이러한 문제. 이 방법은 강력한 기능, 생화학 적 연구 4 충분한 수율을 제공하고, 어떤 경우에는, 추가의 정제없이 결정화 의무가 단백질을 초래한다. 이 프로토콜은 표현을 극대화하고 정지에서 재배 인간 배아 신장 (HEK)에서 분비 포유류 단백질 293F 세포 산출하기 위해 여러 가지 기술에 대해 설명합니다. 형질 전환 효율 (비용), 단백질 생산 및 정제 모두 크게이 프로토콜에 따라 향상됩니다. 단일 단계의 개환 반응을 통해 카바 메이트의 추가에 의해 변형 된 PEI는 (PEI-TMC-25, REF 5에 상세히 설명 합성 및 특성)을 크게, 트랜 스펙 션 효율을 향상 양이온으로부터 세포 독성을 줄여따라서 막 중단 및 실험 비용을 절감 할 수 있습니다. 또한, 세포 생존 및 단백질 발현이 크게 포도당과 비타민 공급 배양 보조제의 첨가로 향상된다. 중요한 kifunensine와 당화 단백질, 치료의 제조, 만노 I의 비 독성 화학 억제제, 대신에 단일 N 아세틸 글루코사민으로 단백질을 수득 엔도 글리코시다 제의 EndoHf에 의해 제거 될 수있다 정의 미성숙 글리 칸으로 단백질을 생산 전체 길이가 6 글리 칸을 N- 결합. 마지막으로, 혈청, 화학적 매체에 단백질의 분비는 구조적, 생화학 적 연구에 대한 신속하고 손쉬운 정화를 할 수 있습니다. 단일 단계 니켈 니트릴 로트리 아세트산 (NTA 니켈) 수지의 정제는 경우에 따라, 결정화를 위해 충분한 순도의 단백질을 얻을 수 있고, 상징액의 종 및 오염의 대부분을 제거한다.

<table>
	<tbody>
		<tr>
			<td>Culture Flasks</td>
			<td>GeneMate</td>
			<td>F-5909B</td>
			<td></td>
		</tr>
		<tr>
			<td>293 Freestyle Media</td>
			<td>Gibco/Life Technologies</td>
			<td>12338-018</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco/Life Technologies</td>
			<td>35050-061</td>
			<td>Use in place of Glutamine</td>
		</tr>
		<tr>
			<td>Hype 5 transfection reagent</td>
			<td>Oz Biosciences</td>
			<td>HY01500</td>
			<td></td>
		</tr>
		<tr>
			<td>293fectin transfection reagent</td>
			<td>Life Technologies</td>
			<td>12347019</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI transfection reagent</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow&#160;</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo Hf</td>
			<td>NEB</td>
			<td>P0703L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose Resin</td>
			<td>NEB</td>
			<td>E8021S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Boost R05.2</td>
			<td>HyClone</td>
			<td>SH30584.02</td>
			<td>Cell Culture Supplement</td>
		</tr>
		<tr>
			<td>GlucCell</td>
			<td>CESCO Bioengineering</td>
			<td>DG2032</td>
			<td>Glucose Monitoring System</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>519850.91</td>
			<td>Serum Free Medium for DNA transfection</td>
		</tr>
		<tr>
			<td>Luria Broth (LB Media)</td>
			<td>Life Technologies</td>
			<td>10855-001</td>
			<td></td>
		</tr>
		<tr>
			<td>GC10 Competent Cells</td>
			<td>Sigma-Aldrich</td>
			<td>G2919</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

본원에서 골수 세포를 2 (hTREM2 잔사 19부터 1백32까지)에 발현 인간 단백질 트리거링 수용체로부터 분비되는 13 kDa의 면역 글로빈의 (IG)에인가 도메인이 발현 시스템의 결과에 따른다. TREM2 두 개의 이황화 결합 및 N - 결합 글리코 실화 부위를 갖는 단일 IG 세포 외 도메인을 함유하는 I 형 막 횡단 단백질이다. 다른 많은 IG 도메인 단백질 (8)과는 달리, TREM2 세균 봉입체 (9)에서 접힘 의?...

Watch this Scientific Journal Video about Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization at JoVE.com

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

효율적인 포유 동물 세포 발현 및 결정화에 대한 세포 외 당 단백질의 단일 단계 정화

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

efficient-mammalian-cell-expression-single-step-purification

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Biology

13.7K 조회수.  Washington University School of Medicine. This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

비디오: Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

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 ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

포유 동물 세포에 Glycoproteins의 N - 연결 설탕 체인 펄스 체이스 분석

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

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 ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

포유류의 링커 - 히스톤 Subtypes의 표현 분석

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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 ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...