This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

발현 플라스미드 pHLsec-GZM 1. 생산 <ol><li> 인간 NK 세포로부터 manufacturer` 다음 적합한 RNA 분리 방법을 이용하여 첫 번째 가닥의 cDNA를 합성 키트를 사용하여 스크립트 작성 역방향 (26 또는 다섯 인간 Gzms 발현 NK 세포주 NK-92에서와 같이 제조 차 전지), 총 RNA를 준비 의 권장 사항. 23 (라두 Aricescu과 이본 존스, 옥스포드, 영국의 대학 종류의 선물)에 설명 된대로 GZM cDNA를 AgeI과 KpnI으로 절단 부위 (그림 1)를 사용하여 pHLsec으로 PCR 및 복제를 사용하여 증폭. 참고 : 표 1의 다음 프라이머를 사용합니다. </li><li> 순서에 의해 올바른 삽입을 확인합니다. DH5α 세포에서 발현 플라스미드를 확장하고 독소가없는 플라스미드 분리 키트를 사용하여 정화하고 제조업체의 지침을 따르십시오. </li><li> 된 unti -20 ° C에서 2 ㎎ / ㎖ 저장의 농도로 내 독소가없는 멸균 수에 정제 된 플라스미드를 재현 탁L 사용. </li></ol> HEK293T 세포에서 Gzms 2. 표현 <ol><li> 시약의 조제 <ol><li> 표준 배지를 준비합니다. 둘 베코 변형 이글 중간 (DMEM)를 함유하는 고 글루코스 (25 mM)을 루타 (4 mM)을 위해, 피루브산 나트륨 (1 mM)을 (표준 소 태아 혈청 (FCS), 페니실린 (100 단위 / ml), 스트렙토 마이신의 10 %를 추가 100 μg의 / ㎖). </li><li> 형질 매체를 준비합니다. 페니실린 - 스트렙토 마이신이없는 배지에 25 μg의 / ㎖ 클로로퀸 (-20 ℃에서 보관 PBS에서 1000 배 주식에서 형질의 날에 갓 추가)를 추가합니다. </li><li> 무 혈청 배지를 준비 : 무 혈청 배지에 HEK293 세포 글루타민 (4 mM)을, 페니실린 (100 단위 / ml), 스트렙토 마이신 (100 μg의 / ㎖) 및 2.5 ㎎ / L의 암포 테리 B. 추가하세요 </li><li> 사용하기 전에 0.45 μm의 필터를 표 2와 필터에 설명 된 솔루션을 준비합니다 : </li></ol></li><li> HEK293T 세포 확대 <ol><li> 20 ㎖ 숭배에 HEK293T 세포를 성장150 X 25mm 조직 배양 접시를 사용하여 URE 매체. </li><li> 80 %의 포화 상태에서 분할 세포 (1 분할 비율 : 4, 일반적으로 매 3 일째). 세포는 느슨하게 그들은 기계적으로 엄격로 pipetting 아래로 트립신하지 않고 분리 할 수​​ 있습니다 연결합니다. </li><li> 플레이트 세포 형질 감염 전날은 형질 감염 (시딩 밀도 ~ 2 × 105 세포 / ml)의 날 60~70%의 생장을 얻었다. 평소 준비 크기는 20-25 배양 접시로 구성되어 있습니다. </li></ol></li><li> 칼슘 - 포스페이트 형질 <ol><li> 형질 감염 전에, 1 시간에 20 ml의 형질 감염 배지로 표준 배지를 변경. 이 1 시간 배양 단계의 끝 부분, 10.95 ML의 DDH 2 0 50 ml의 튜브 2M 염화칼슘 2의 1.55 ㎖ (1 관 / 5 요리)와 플라스미드 DNA의 400 μg의 혼합. </li><li> (1) 형질 전환 이전에 최소 2, DNA-칼슘 용액 12.5 ml의 12.5 ml의 2 배 HBS를 추가 튜브의 반전에 의해 혼합하고 실온에서 30 초 동안 품어. </li><li> 일 추가E 혼합물을 직접 세포 (접시 당 5 ㎖)에 적가 매체에 2.3.2에서 제조. 약간 오렌지 색상으로 매체를 회전, 전체 영역에 걸쳐 고르게 뿌린다. </li><li> 조직 문화 인큐베이터에서 7-11 시간 (37 ℃, 5 % 가습 공기 중 CO 2)의 배양 접시를 품어. 배양 후, 미세한 침전물을 볼 수 있습니다. 미리 예열 PBS로 조심스럽게 씻어, 형질 매체를 제거하고 2.1.3에서 제조 된 20 ml의 무 혈청 배지를 추가합니다. 조직 문화 인큐베이터에서 72 시간 동안 품어. </li><li> SDS-PAGE에 세포 상층 액의 샘플 (~ 20 μL)를 실행하고, 그랜 자임 밴드를 시각화 쿠마 브릴리언트 블루로 염색하여 형질 전환 및 발현 효율을 분석 할 수 있습니다. </li></ol></li><li> 니켈 IMAC에 의해 배양 상층 액에서 Gzms의 정제 <ol><li> 배양 후, 250 ml의 튜브에 세포 배양 상층 액을 가만히 따르다 및 원심 분리하여 취소합니다. 첫 번째 스핀을 수행 (400 XG, 10 분, 4 °분리 된 세포에서 매체를 취소합니다 C). 남아있는 세포 파편을 제거하기 위해 4 ° C에서 4,000 XG, 30 분에서 신선한 250 ml의 튜브와 스핀에 뜨는을 전송합니다. </li><li> 5 M NaCl을 5 ㎖, 삭제 상층 액 250 ml의 당 0.25 M 니소 (4) 6.25 2 M 트리스 자료 (PH 8) ml의 1 ML을 추가합니다. 500㎖의 진공 필터 유닛 (0.45 mm)를 사용하여 상청액을 필터. </li><li> 적절한 FPLC 시스템을 사용하여 그분의 결합 버퍼를 (10 열 볼륨, CV)와 니켈 - 아이맥 열 평형. </li><li> 4 ℃에서 0.5 ㎖ / 분의 유속으로 칼럼을 평형화 클리어 상등액을 적용한다. </li><li> 상등액을 도포 한 후 UV 흡광도가 기준선 (통상 10 CV)에 도달 할 때까지, 그의 결합 완충액으로 칼럼을 세척 하였다. </li><li> 용출 Gzms 0.5 ml의 유속으로 20 ㎖의 선형 구배 (0 ~ 250 mM의 이미 다졸)로 / 분 ML-2 분획을 수집한다. SDS-PAGE 및 쿠마시 염색으로 용출 분획을 분석한다. </li></ol></li><li> 엔테로 키나제 (EK)치료 및 양이온 교환 크로마토 그래피에 의한 최종 정리 <ol><li> ≤10와 MWCO 투석 튜브 함유 분획을 풀 GZM. 사전 EK 제어와 같은 -20 ° C에서 작은 샘플을 저장합니다. </li><li> 투석 튜브에서 직접 풀링 부분에 0.02 단위 / 초기 상층 액 50 ㎖에 EK을 추가하고 O / N EK-버퍼에 실온에서 (최소 16 시간) (4 L, 한 번 변화 버퍼를) 투석. </li><li> 다음날, EK는 SDS-PAGE 및 쿠마시 염색으로 사전 EK 제어에 비해 Gzms 처리 분석. </li><li> N 말단 처리가 완료되면 (4 L)를 버퍼 S 투석 완충액을 변경하고 다른 RT에서 4 시간 동안 투석. 필터 투석​​액 (0.45 μm의). </li><li> 4 ℃에서 0.5 ㎖ / 분의 유속으로 칼럼에 샘플을로드 S 버퍼 A. 가진 S-열 평형. </li><li> UV 흡광도 기준 (약 10 이력서)에 도달 할 때까지 샘플로드 한 후, S 버퍼와 열을 씻는다. 30 ml의 선형 구배 (150 mM의 NaCl을 1000)와 Gzms을 용출. GzmA 전자~ 750 mM의 염화나트륨에서 ~ 700 mM의 염화나트륨과 GzmM에서 ~ 650 mM의 NaCl을, GzmB에서 류트. </li><li> SDS-PAGE 및 비색 분석 (아래 참조)에 의한 용출 분수를 분석합니다. Gzms과 농축액을 함유하는 분획을 풀 (약 30 배 이상, 약 100 μM의 농도로)에서의 스핀 필터 (15 ㎖, 10 MWCO). -80 ° C에서 집중 GZM 준비 및 저장 나누어지는. </li></ol></li></ol> 3. 테스트 GZM 활동 <ol><li> 시약의 조제 <ol><li> 50 mM 트리스 - 자료, pH가 7 : 비색 분석 버퍼를 준비합니다. <ol><li> GzmA의 측정을 위해, 0.2 mM 내지 5,5&#39;- 디티 오 - 비스 (2- 니트로 벤조산)의 최종 농도로 검정 완충액에 나트륨-CBZ-L- 라이신을 thiobenzyl 에스테르 (S-BZL) (BLT)를 추가 0.22 mm의 농도 (DTNB). GzmB를 들어, 0.2 mM의 BOC-알라 - 알라 -의 ASP-S-BZL (AAD) 및 0.22 mM의 DTNB을 포함한다. GzmM 들어, 0.2 mM의 SUC 닐 -Ala-Ala-Pro-의 Leu-35㎕를 PNA () (AAPL)를 포함한다. 합성 펩티드는 DMSO에 (적절한 재고 솔루션에서 추가, STOR에드 갓 분석하기 전에 버퍼에 -20 ℃)​​에서. </li></ol></li><li> 절단 분석 완충액 준비 : 100 mM의 염화나트륨, 50 mM 트리스 - 자료, pH가 7.5 </li></ol></li><li> 비색 분석 <ol><li> 96 웰 평면 바닥 판에서 용출 분수에서 작은 GZM 샘플 (&lt;5 μL) 또는 집중 주식을 배포합니다. 양성 대조군 (GZM 시험 제제 또는 조질 NK 세포 용 해물) 및 음성 대조군 (전용의 버퍼)를 포함한다. </li><li> (멀티 채널 피펫을 사용하여) 각 웰에 분석 완충액 100 μl를 추가합니다. 37 ℃에서 10 분 동안 품어. 마이크로 플레이트 리더에서 405 nm에서 OD를 측정한다. </li></ol></li><li> 분열 분석 <ol><li> 희석액과 - 정제 된 기판을 사용하여 절단 분석법 (효율적인 GZM 기판의 몇 가지 예는이 섹션의 끝에 결과 섹션과 주에 제시 400 nM의 네이티브 또는 재조합, 100)을 단백질 기질을 공동 배양 GZM의 다양한 시간에 대한 분석 버퍼 (400 nm에서 시작).SDS-PAGE 및 쿠마 또는 실버 염색으로 분석; 또는 웨스턴하여 특정 항체를 사용하여 블 롯팅. </li><li> 세포 용 해물을 사용하여 절단 분석법, 37 ° C 세번에서 에탄올 / 드라이 아이스 배스 융해에서 동결 세이 버퍼를 Lyse 1 ㎖에서 106 HeLa 세포 (또는 가능한 종양 세포주)를 일시. (4 ° C에서 10 분 동안 15,000 XG)을 원심 분리하여 세포 파편을 제거합니다. </li><li> 위와 같이 다양한 농도와 시간에 Gzms와 클리어 해물을 공동-품어. 쪼개짐은 적절한 항체를 사용하여 면역 블에 의해 검출된다. 참고 : 선의의 기판의 몇 가지 예하는 상업 항체가 사용할 수 있습니다 입찰 (BH3-상호 작용 도메인 죽음 작용제)와 GzmB 27,28에 의해 절단 카스파 제 3; GzmA (29)에 의해 절단 여러 이기종 핵 리보 (hnRNPA1, A2 및 U); GzmM (30)에 의해 거대 세포 바이러스 인 단백질 (71). </li></ol></li></ol>

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The authors have nothing to disclose.

GzmA 및 GzmB의 고전과 광범위하게 연구 역할은 기공 형성 단백질 PFN 1로 그들의 세포질 배달 후 포유 동물 세포에서 세포 사멸의 유도이다. 최근 Gzms의 세포 독성 스펙트럼 세균 9,10뿐만 아니라 특정 기생충 (11)에 포유류 세포에서 현저하게 확대시켰다. 또한, GzmA 및 GzmB의 비 고전적, 세포 외 기능뿐만 아니라 다양한 고아 Gzms의 생물학적 중요성은 여전히​​ 불분명하다. 이 ...

Gzms 전문 CTL의 리소좀과 NK 세포 1 지역화 높은 상동 세린 프로테아제의 가족입니다. 이러한 킬러 세포의 세포 독성 과립은 제거 2,3 향하는 표적 세포의 인식에 Gzms와 동시에 출시되는 막 방해 단백질 PFN 및 GNLY가 포함되어 있습니다. (- G, K, M과 N GzmA) 인간 (GzmA, B, H, K와 M)에서 다섯 Gzms 및 마우스에서 10 Gzms이 있습니다. GzmA과 GzmB이 가장 풍부하고 광범위하게 인간과 생쥐 한 연구이다. 그러나, 최근의 연구들은 세포 사멸 경로뿐만 아니라 건강과 질환 4 다른, 소위 고아 Gzms에 의해 매개되는 생물학적 효과를 추가로 조사하기 시작했다. 5,6- PFN에 의해 표적 세포 내로 전달 된 경우 Gzms의 가장 잘 알려진 함수와 GzmA GzmB의 특히 포유 동물 세포에서 프로그래밍 된 세포 사멸을 유도한다. 그러나더 최근의 연구는 또한 PFN 7,8 독립적으로 세포질 전달, 면역 조절 및 염증에 깊은 영향을 Gzms의 세포 외 효과를 보여 주었다. Gzms의 세포질 기입 한 후 효율적으로 살해 세포의 스펙트럼은 최근 박테리아 9, 10, 심지어 특정 기생충 (11) 포유 동물 세포에서 확대되었다. 이러한 최근의 발견은 GZM 연구원에 대한 완전히 새로운 분야를 열었다. 따라서, 비용 효율적인, 고 수율 포유 동물 발현 시스템은 크게 그 미래 연구에 대한 방법을 완화합니다. 네이티브 인간, 마우스 및 래트 Gzms 성공적 CTL 및 NK 세포 라인 12-14의 과립 분획으로부터 정제 하였다. 그러나, 우리의 손에 이러한 정제 기술의 수율은 0.1 ㎎ / ℓ, 세포 배양 (미공개 관측 및 12)의 범위이다. 또한, 인접하여 오염이없는 단일의 GZM 크로마토 해상도R Gzms 및 / 또는 과립 형태로도 존재하는 단백질 (게시되지 않은 데이터와 12, 14)에 도전하고있다. 재조합 Gzms 심지어 이러한 HEK 293 18,19 같은 포유 동물 세포에서 세균 15, 16, 효모, 곤충 세포 및 17에서 제조 하였다. 만 포유류 발현 시스템은 원시 세포 독성 단백질과 동일한 번역 후 변형으로 재조합 효소를 생산할 수있는 가능성을 부담. 번역 후 변형은 엔도 사이토 시스에 의해 특정 흡수 및 표적 세포 20-22 내의 단백질 분해 효소의 세포 내 현지화에 연루되어있다. 따라서, pHLsec (23)를 사용하여 GZM 발현 플라스미드 백본 (라두 Aricescu과 이본 존스, 옥스포드, 영국의 대학의 일종 선물), 우리는 높은 수율 단백질 생산을위한 간단하고, 시간과 비용 효율적인 시스템을 구축 HEK293T 세포. pHLsec는 닭 Β-액틴 프로모터와 CMV 증강을 결합; 함께, 이러한 요소는 demonstra테드 다양한 세포 라인 (24)에서 가장 강력한 프로모터 활성. 또한, 플라스미드는 토끼 Β-글로빈 인트론, 최적화 된 코작과 분비 신호리스 - 6xHis 태그 및 폴리 신호가 포함되어 있습니다. 삽입이 분비 신호와 적절한 N- 말단 도메인을 부족한 단백질에 대한 최적의 발현과 분비 효율을 보장리스 - 6xHis 태그 (그림 1) 사이에 편리하게 복제 할 수 있습니다. Gzms의 발현을 위해, 우리는되도록 EK 치료 분비 Gzms는 (활성 Gzms는 N - 말단으로 시작 활성화 된 엔테로 키나제 (EK) 사이트 (DDDDK) 뒤에 벡터에 의해 제공 분비 신호 내인성 분비 신호 서열로 대체 아미노산 서열 IIGG 25). 또한이 방법에 대한 찬성, HEK293T 세포는 둘 베코의 수정 이글의 중간 (DMEM)로, 저가의 매체에 빠른 속도로 성장하고, 비용 효율적인 칼슘 포스페이트 형질 전환 방법에 적합하다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

다음 섹션에서 우리는 방법을 조명 GzmA 준비의 전체​​ 문서를 발표 할 예정이다. 또한 성공적으로 정화 효율 및 작동에 대해서는 유사한 결과를 생성하고 GzmB GzmM 정제. 그러나, 그 후 준비에서 우리는 데이터의 몇 선택한 조각을 보여줍니다. 현재 프로토콜 다음 293T 세포로부터 GzmB 제제는 다양한 생물학적 분석 시스템 9,29,31-34 그들의 활동을 강조 여러 발표 된 연구에 사용 하였다. <p...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

포유 동물 세포에서 인간의 Granzymes의 높은 수율과 비용 효율적인 발현 시스템

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

a-high-yield-cost-efficient-expression-system-human-granzymes

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JoVE Journal

Biology

9.7K 조회수.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 

비디오: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

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

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

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 ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

파우더 샘플의 절대 양자 수율 측정

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

포유 동물 세포의 세포주기의 위치 분석

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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 ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

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 ...