이 작품은과 ShanghaiTech 대학, 상하이, 중국 (DP1CA174426 카를로스 F. 바스 (Barbas)로) (JL에) 건강의 국립 연구소에 의해 지원되었다. 분자 그래픽 파이 몰을 이용하여 생성 하였다.

1. 복제 <ol><li> 애완 동물-28 발현 벡터 시스템에 클로닝 SUB 요청 (PET-2F-ZIF)에 따라 사용할 수 있습니다되었습니다 알라닌 치환 두 손가락 ZIF 도메인을 가져옵니다. (34) </li><li> PCR은 프라이머 플라스미드 에메랄드 pBAD에서 EmGFP를 증폭 5 &#39;XmaI-EmGFP (5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; 굵게 XMA I 사이트) 및 3 &#39;를 SacI-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ; 굵게 골목 I 사이트). <ol><li> 주형 DNA 5 ng를 10 배 중합 효소 완충액 10 μL, 증류 / 탈 이온수로 이루어진 하나의 Taq DNA 중합 효소, 0.2mM의 각각의 dNTP의 단위 (U)와 나머지 부피 100 μL 용액에서 각 프라이머의 0.2 μM 사용 . 사이클링 조건을 사용하여 5 분 동안 95 ° C를; 30 초 동안 95 ° C의 30주기, 30 초 동안 55 ° C에서 1 분 72 ° C; 10 분 동안 72 ° C에서. 겔 extracti 의해 PCR 생성물을 정제하여에와 애비 260 × 50 ㎍ / ml의 측정 분광 광도계를 사용하여 DNA 농도를 결정한다. </li></ol></li><li> 다이제스트 애완 동물 2F-ZIF 및 제한 EmGFP을 코딩하는 삽입 DNA의 1 μg의 당 효소 10 U를 사용하여 37 ° C에서 3 시간 동안 권장 버퍼에 XMA I과 골목 I 효소. 이러한 티듐 브로마이드 인터 형광 염료를 이용한 아가로 오스 겔 전기 영동에 의해 DNA를 시각화. </li><li> 젤 추출 키트에 의해 소화 DNA를 정화하고 복근 260 × 50 ㎍ / ml의 측정 분광 광도계에 의해 DNA 농도를 결정한다. </li><li> 실온에서 최소 1 시간 동안 T4 DNA 리가 제 1 U를 사용하여 애완 동물 2F-ZIF의 50 ~ 100 NG로 정제 EmGFP 인코딩 DNA를 결찰. 인서트 한 투 벡터 몰비 : 최상의 결과를 위해,도 6을 사용하여 연결 반응을 수행한다. </li><li> 얼음에 어떤 화학적으로 유능한 XL-1 블루 대장균 세포의 50 μl를 해동 및 10 ~ 20 ng의 결찰 애완 동물 2F-ZIF-EmGFP의 가볍게 섞는다. </li><li> 3 얼음에 보관0 분. 열 충격 90 초 동안 42 ° C에서 혼합물은 진탕 37 ° C에서 1 시간 동안 Catabolite 탄압 (SOC) 슈퍼 최적 배지 2 ml의 세포를 복구 할 수 있습니다. </li><li> 50 ㎍ / ㎖의 카나마이신과 원성 국물 (LB) 한천 접시에 복구 문화의 100 μl를 확산하고 37 ° C에서 O / N을 배양한다. </li><li> 다음 날, 슈퍼 국물 (SB) 또는 50 ㎍ / ㎖의 카나마이신 37 ° C에서 LB 한천 플레이트와 문화 O / N에서 한 식민지 포함하는 LB 문화의 6 ml에 접종. 주 : 콜로니 PCR 프라이머는 5 &#39;XmaI-EmGFP 및 3&#39;SacI로-EmGFP 미니 프랩을 사용하는 것은 이전에 양성 클론을 식별하는데 사용될 수있다. </li><li> 미니 프렙에 의하여 애완 동물-2F-ZIF-EmGFP을 정화하고 T7 프로모터를 사용하여 DNA 염기 서열에 의해 플라스미드를 확인 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;). </li></ol> 2. 발현 및 정제 <ol><li> 화학적으로 유능한 BL21 E.의 50 μl를 해동 얼음에 대장균 세포와 PL 애완 동물 2F-ZIF-EmGFP 100 NG로 가볍게 혼합asmid. 단계 1.7-1.8에 설명 된대로 변환. </li><li> 다음날, 단일 콜로니로 50 ㎍ / ㎖의 카나마이신을 함유하는 LB 배지 10 ㎖에 접종하고 진탕하면서 37 ℃에서 O / N 성장. </li><li> 다음 날, 50 ㎍ / ㎖의 카나마이신, 0.2 % 글루코스 및 100 μM ZnCl이 보충 된 LB 배지 1 L로 O / N 배양의 10 mL로 희석. 0.8의 600 nm의 (OD 600)에서 광학 밀도에 진탕 37 ° C에서 문화를 성장과 2 mM의 이소 프로필-β-D-1-D- 티오 갈 락토 피 라노 시드 (IPTG)와 단백질 발현을 유도한다. 4 ° C에서 10 분 동안 5,000 XG에 원심 분리하여 37 ° C에서 성장의 6 시간, 수확 세포 후. 참고 : 유도 조건이 매우 다양하고 표현되는 단백질의 안정성에 따라 달라집니다. 0.8 (600)에 도달 할 때까지 OD OD 600 매 30 분을 모니터링합니다. </li><li> 용해 완충액 5 ㎖ (50 mM의 트리스 -HCl, 500 mM의 염화나트륨에서 세포 펠렛을 재현 탁, 100 μM ZnCl 2, 1mM 디티 오 트레이 톨 (DTT), 1 ㎜의 MgCl 2, 1 mM의 페닐 메틸 설 포닐 플루오 라이드 (PMSF) 및 10 mM의 이미 다졸, pH를 8.0). 다음과 같은 설정으로 초음파에 의해 얼음에로 용해는 세포 : 50 % 전원 출력 / 10 초 오프 간격으로 5 초와 4 분의 처리 시간. 솔루션을 과열하지 마십시오. </li><li> 4 ° C에서 30 분 동안 25,000 XG에서 세포 용 해물을 원심 분리기와 신선한 수집 관에 뜨는을 전송합니다. 최상의 결과를 위해, 4 ° C에서 다음의 모든 단계를 수행합니다. </li><li> 열 평형의 Ni-NTA 슬러리의 1 ml로 미리 포장을 통해 상층 액을 실행합니다. 세척 버퍼의 20 ㎖ (50 mM 트리스 - 염산, 500 mM의 NaCl을, 100 μM ZnCl 2, 1 mM의 DTT, 1 ㎜의 MgCl 2, 30 mM의 이미 다졸, 산도 8.0)와 수지 씻으십시오. </li><li> 5 용출 완충제 ㎖ (50 mM의 트리스 -HCl, 500 mM의 NaCl을 100 μM ZnCl 2, 1 mM의 DTT, 1 ㎜의 MgCl 2, 300 mM의 이미 다졸, pH를 8.0)으로 용출시켰다. </li><li> 버퍼 저장 버퍼와 용출 된 단백질 (50 mM의 교환트리스 -HCl, 500 mM의 NaCl을 100 μM ZnCl 2, 1 mM의 DTT, 1 ㎜의 MgCl 2, 10 % 글리세롤, pH를 8.0) 및 제조자의 지시에 따라 원심 분리하여 회전 농축기를 이용하여 적어도 40 μM로 단백질을 농축시켰다. </li><li> BCA 또는 브래드 포드 분석에 의해 단백질 농도를 결정합니다. 10 분 동안 95 ° C에서 2 μL 2 × SDS-PAGE 로딩 염료, 종기와 정제 된 단백질의 2 μl를 혼합 한 후 4 %에 단백질 순도 (그림 2) 평가 20 % 트리스 - 글리신 SDS-PAGE를 해결. </li></ol> 3. 단백질 저장 <ol><li> -80 ° C에서 액체 질소와 저장소에 농축 된 단백질, 플래시 동결 나누어지는. EmGFP 융합 단백질, 단백질 빛으로부터 보호하기 위해 알루미늄 호일로 커버 튜브. </li><li> 반복 동결 단백질 용액의 해동을 피하십시오. 참고 : ZIF - 융합 단백질이 1 개월 이상 이러한 조건에서 안정하다. 부적절한 또는&gt; 4개월 스토리지 될 수 있습니다단백질 침전 또는 EmGFP의 광표백에. </li></ol> 4. 단백질 전달 <ol><li> 10 %를 함유하는 둘 베코 변형 이글 배지 (DMEM)에서 HeLa 세포를 유지 (v / v)의 소 태아 혈청 (FBS), 1 % 항생제 - 항진균제 5 % CO 2와 완전히 가습 분위기에서 37 ℃. 참고 : 세포는 30 개 이상의 시간이 단백질 전달하지 않는 것이 좋습니다 계대. </li><li> 사전 코트 25 ° C에서 30 분 60에 대한 폴리 라이신의 50 μg의 / ㎖의 500 μL와 24 웰 플레이트. 웰 당 2 × 105 세포의 밀도로 24 웰 플레이트 상에 종자 세포. 세포가 80 % 사이에 -90 %의 합류 일단 파종 후 24 시간에서, 또는, 각 웰에서 미디어를 제거하고 예열 된 무 혈청 DMEM 500 μL (SFM)로 세척한다. </li><li> 각 웰에, SFM의 250 μL가 ZIF-EmGFP 단백질의 2 μM, 100 μM ZnCl 2를 포함하는 추가합니다. 1 시간 동안 37 ℃에서 세포를 배양한다. 참고 : ZIF 도메인은 세포를 입력 홍보imarily 에너지에 의존하는 메커니즘입니다 macropinocytosis, (34)를 통해. 따라서, 단백질의 효율적인 세포 내재화 37 ° C에서 배양해야한다. </li><li> 칼슘과 마그네슘이없는 500 μL로 3 회 세포에서 미디어를 제거하고 세척 둘 베코의 인산염 완충 식염수 헤파린의 0.5 ㎎ / ㎖로 보충 (DPBS). 참고 : 헤파린은 다운 스트림 분석을 복잡하게 할 수 표면에 결합 된 단백질을 제거 할 필요가있다. </li><li> 향상된 배달 3 회까지 (선택 사항) 반복 치료. </li><li> 바로 2 분 37 ° C에서 0.05 % 트립신 EDTA와 소화에 의해 세척 헤파린 후 처리 된 세포를 분리합니다. 1 % FBS가 보충 된 0.5 ㎖의 DPBS에서 분리 된 세포를 다시 일시. </li><li> 형광 염료 (FITC)의 채널 (도 3)을 이용하여 유세포 분석기 (35)에 의해 각 시료의 형광 강도를 측정한다. 의 인구를 배치 앞으로 분산 형 (FSC)와 측면 산란 (SSC)를 조정서로 다른 특성을 가진 집단이 서로 해결을 보장 규모에 관심. </li><li> 각 샘플에 대한 10,000 라이브 이벤트를 수집 및 처리 만 SFM 해당 셀에 ZIF-EmGFP로 처리 된 HeLa 세포로부터의 데이터 분석 소프트웨어. 36 정규화 유세포 형광을 사용하여 데이터를 분석한다. </li></ol>

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Biol. 400, 96-107 (2010).</li><li>Gaj, T., Guo, J., Kato, Y., Sirk, S. J., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+knockout+by+direct+delivery+of+zinc-finger+nuclease+proteins.">Targeted gene knockout by direct delivery of zinc-finger nuclease proteins.</a> Nat. Methods. 9, 805-807 (2012).</li><li>Gersbach, C. A., Gaj, T., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+zinc+finger+proteins:+the+advent+of+targeted+gene+regulation+and+genome+modification+technologies.">Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.</a> Acc. Chem. Res. 47, 2309-2318 (2014).</li><li>Gaj, T., Gersbach, C. A., Barbas, C. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+class+of+human+proteins+that+deliver+functional+proteins+into+mammalian+cells+in+vitro+and+in+vivo.">A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo.</a> Chem. Biol. 18, 833-838 (2011).</li><li>Gaj, T., Liu, J., Anderson, K. E., Sirk, S. J., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+Cys2-His2+zinc-finger+domains.">Protein delivery using Cys2-His2 zinc-finger domains.</a> ACS Chem. Biol. 9, 1662-1667 (2014).</li><li>Radcliff, G., Jaroszeski, M. 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저자가 공개하는 게 없다.

여기, 세포 투과성 아연 손가락 (ZIF) 도메인을 사용하여 단백질 전달을위한 단계별 프로토콜이 제공됩니다. ZIF 도메인 융합 효소화물 (34)의 활성을 감소시키지 않는다; 생산 및 수정되지 않은 단백질과 관찰 된 것과 거의 동일한 수익률 단백질의 정제 할 수 있습니다; 전통적인 세포 투과 펩티드 또는 단백질 전달체 시스템 효율성을 초과하여 세포 유형 내로 다양한 단백질 및 효소를 운반 할 수있다. 함께, 이러한 연구 결과는 다양한 유형의 애플리케이션에 직접 단백질을 세포 내로 전달을 매개하는 ZIF 도메인 넓은 전위를 나타낸다. 최대 단백질 전달은 이전보다 양전하를 운반 3-4 손가락 ZIF 도메인의 배열을 연장하는 사실에도 불구하고, 오직 두 손가락 ZIF 도메인을 사용하여 달성 하였다. 이러한 결과는 세포 - 매개 ZIF 항목 전하, INC 이외의 요인에 의해 영향을받을 수 있음을 나타낸다단백질 안정성 또는 구조적 강성을 luding. ZIF 도메인 매개 단백질 전달은 에너지에 의존하는 것으로 밝혀 따라서 단백질로 치료 모든 세포가 37 ° C에서 배양 할 것을 요구했다. 다양한 세포 내 이입 경로, macropinocytosis의 소분자 저해제의 사용을 통해, 그리고 낮은 정도의 카베 올린 의존적 세포 내 이입에 ZIF - 매개 세포 항목의 주요 경로 인 것으로 결정 하였다. (34) 특히, 다른 단백질 전달 시스템과 달리, ZIF 도메인은 효율적으로 융합 된 거대 분자의 세포 내 전달화물의 상부를 중재하는 엔도 좀을 빠져 강력한 단백질 전달을 달성하기위한이 영역의 전위를 강조 할. 우리의 경험에 의하면, 세포의 파종 밀도는 단백질 전달의 높은 수준을 달성하기위한 중요한 공정이다. 우리는 그들이 80 % -90 %의 포화 상태에 도달하면 세포 치료 이전에 관찰하는 것이 좋습니다&gt; 95 % 컨 플루 쇼 하위에 시드 셀최적의 전달 용량, 낮은 밀도 (&lt;50 %)로 접종 세포 독성 단백질 유발되기 쉽다있다. 중요한 것은, 높은 분리 경향과 세포 유형, 세포 배양 플레이트 폴리 라이신과 사전 코팅을 권장합니다. 폴리 라이신은 음전하를 세포 표면의 구성 요소와 정전 기적 상호 작용을 통해 세포 부착을 용이하게한다. 각 ZIF 도메인의 α 나선 DNA 결합 잔류 물 DNA 인식에 대한 가능성을 제거하기 위해 제거되었지만, ΒΒα ZIF 도메인 배를 안정시키기 위해 아연 2+ 이온 좌표 시스테인과 히스티딘 잔류 물은 그대로 유지됩니다. 따라서, 우리는 임의의 저장 버퍼 단백질 무결성을 유지 ZnCl 2의 적어도 100 μM로 보충 할 것을 권장합니다. ZIF 도메인이 이전에 다양한 종류의 세포에 단백질 및 효소를 제공하도록 도시되​​었지만, ZIF 전달의 효율도 macromo 모두에 의존 할 수도lecular화물 및 단백질 농도. 예를 들어, 세포는 두 손가락 ZIF-루시페라아제 융합 단백질을 처리 한 세포가 두 손가락으로 처리하면서, 더 높은 농도의 활성 감소로, 0.5 μM 단백질로 처리 할 때 최대 발광을 표시하는 관찰 EmGFP는 형광의 용량 의존적 증가를 나타내 강도 8.0 μm의 단백질까지, 연속 단백질 치료시. 따라서 우리는 농도 범위에 걸쳐 각 고유 ZIF 도메인 융합 세포 침투 능력을 평가 추천. 아직 증명되지 않았지만 마지막으로, 우리는 ZIF 도메인 전달이 세포 내로 거대 분자의 다양한 어레이를 제공 할 수있는 매우 유연한 플랫폼이라고 예상된다. DNA 및 RNA 모두 가수 분해성 화학적 링커를 통하여 ZIF 도메인의 표면에 관능 또는 일시적 ZIF 도메인 캡슐화 세포로 형질 감염 될 때까지 예를 들어, 가능할 수있다. 또한 efficiZIF 단백질 전달 ency 더욱 표면 전하의 최적화에 초점을 합리적 설계 노력에 의해 향상 될 수있다.

고효율 및 다양한 단백질 전달 전략은 많은 기초 연구 및 치료 응용 프로그램에 대한 중요합니다. 세포에 정제 된 단백질의 직접 배달이를 달성하기위한 가장 안전하고 가장 쉬운 방법 중 하나. 1, 2 핵산에서 유전자 발현에 의존하는 전략을 달리 표현, 3-5 단백질 배달에서 삽입 돌연변이 유발의 위험을 제기하지 무관 세포의 전사 / 번역 기계 및 즉각적인 효과를 할 수 있습니다. 그러나 단백질에 세포 침투 활동을 부여하는 대한 단순하고 일반화 방법의 부족은 정기적으로 세포에 직접적인 항목을 혼동. 단백질의 세포 내 전달을 용이하게하기위한 현재의 방법은 천연 또는 6-8 설계된 세포 침투 펩타이드 9-12 과급 전달 도메인, 13, 14 및 15 나노 리포좀 16 바이러스 입자 17,18 발생의 사용에 기초 </SUP&gt;과 고분자 미세 물질. (19) 불행하게도, 이러한 방법의 대부분이 낮은 세포 흡수 속도, 20, 21 가난한 안정성, (22) 실수로 세포 형 특이도, 23 낮은 엔도 좀 탈출 등록 (24)과 또한 독성. (25)에 의해 방해된다, 많은 ​​단백질 전달 기술은 전달 단백질의 생체 활성을 감소시킨다. (14) 우리 연구소는 이전에 아연 손가락 핵산 분해 효소를 증명 (ZFN) 단백질 - 키메라 제한이 프로그램의 Cys 2 - 그의 두 아연 손가락 DNA 결합 단백질과 FokI 제한 엔도 뉴 클레아 제 26 ~ 28의 절단 도메인으로 구성된 엔도 뉴 클레아 제 - 본질적으로 세포 - 아르 투과. (29)이 놀라운 세포 침투 활동은 맞춤 디자인 아연 손가락 도메인, 대상 게놈 엉위한 강력한 도구로 떠오르고있다 DNA 결합 플랫폼의 고유 재산 것으로 나타났다공학에, 30-32 간주은 단백질 표면에 양전하 여섯 잔기의 콘 스텔 레이션의 결과 일 수있다. 실제로, C 6 월 및 N-DEK 비롯한 여러 DNA 결합 단백질이. 세포막을 건너 타고난 능력을 갖는 것으로 밝혀졌다 (33)은보다 최근에, 본 연구실은 이러한 결과를 확장하고 입증하는 아연의 셀 관통 활동 손가락 (ZIF) 도메인은 세포 내 단백질 전달을 위해 활용 될 수있다. 많은 기존의 세포 침투 펩타이드 전달 시스템을 초과 효율성을 빨아들이는 관 주도 특정 단백질화물에 1 또는 2 손가락 ZIF 도메인 중 하나의 유전자 융합. (34) 가장 주목할만한, ZIF 매개 배달 융합 효소화물의 활동을 손상시키고 용이하지 않았다 세포질 전달의 높은 수준. 종합적으로, 이러한 결과는 단백질의 효율적이고 용이 한 전달을 용이하게하기 위해 도메인의 ZIF 가능성을 입증하고, 매크로 잠재적으로보다 다양한 종류의세포에 분자. 여기, 포유 동물 세포에서 단백질 전달을위한 ZIF 기술을 구현하는 방법에 대한 자세한 단계별 프로토콜이 제공됩니다. 우리는 이전의 제품군을 구성 액형, 2 차원, 3 ~, 네살, 다섯 자리와 여섯 손가락 인해 α 나선 DNA 결합 잔류 각각의 대체 선수로, DNA를 결합하는 능력이 부족 ZIF 도메인 만 셀 (34) (도 1)로 단백질을 제공 할 수있다. 두 손가락 ZIF 도메인을 이용하여 HeLa 세포에 GFP 에메랄드 (EmGFP)의 제작 및 형질 도입에 대해서 설명한다. 이 프로토콜은 대장균에서의 발현 및 수용성 거의 모든 포유 동물 세포 유형 가능한 거의 모든 단백질로 확장 가능하다. 예상되는 결과가 제공되며,이 시스템의 성능을 최대화하기위한 전략도 논의된다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

두 손가락 ZIF-EmGFP 융합 단백질은 E.으로 표현 될 수있다 대장균&gt; 95 % 동질성과 높은 수율 (&gt; 25 ㎎ / ㎖) (그림 2). 일반적으로 단일 및 두 손가락으로 ZIF 융합 단백질은 야생형 변성 단백질의 것과 거의 동일한 양으로 제조 할 수있다. 그러나 어떤 상황에서, 다섯 자리와 여섯 손가락 ZIF 융합 단백질은 다운 스트림 응용 프로그램을위한 충분한 높은 수율로 제조 할 수 없습니다. 37 ° C에서 90 분 동안 HeLa 세포 상에 두 개의 손가락 ZIF-EmGFP 단백질의 직접 적용은 EmGFP 형광 (도 3A)에서 용량 의존적으로 증가를 이끈다. 결정적으로, 형광은 ZIF 도메인의 부재하에 관찰되지 않는다. 우리는 이전에 세포의 거의 100 %가 단지 2 μM 두 손가락 ZIF-EmGFP 단백질로 처리 한 후 형광성 관찰하고 ZIF 융합 단백질로 처리 된 HeLa 세포의 단백질 농도에서 EmGFP 형광 양성임을<html> 낮은 0.25 μM (그림 3B). <img alt="그림 1" src="/files/ftp_upload/52814/52814fig1.jpg" /> 그림 1. 구조 및 아연 손가락 단백질의 순서. 하나의 아연 손가락 (ZIF) 도메인의 (상위) 결정 구조. . 아연 2+와 조정 보존의 Cys와 그의 잔류의 측면 체인 이온 스틱 (PDB ID : 2I13)로 표시됩니다 ZIF 도메인의 37 (아래) 순서. 화살표와 실린더는 각각 Β-시트 및 α 나선 차 구조를 나타냅니다. 알라닌으로 치환 된 α 나선 DNA 결합 잔류 물은 분홍색으로 강조 표시됩니다. 세포 내재화을 중재 할 것으로 예상 양전하 잔류 밝은 파란색을 강조하고 있습니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52814/52814fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> &quot;fo를하는 것은 : 유지-together.within 페이지를 =&quot;항상 &quot;&gt; <img alt="그림 2" src="/files/ftp_upload/52814/52814fig2.jpg" /> 원 -, 2 개, 3 개 및 4 개 손가락 ZIF-EmGFP 융합 단백질. ZIF-EmGFP 융합 단백질은 E. 표현했다 깨끗이하게 그림 2. SDS-PAGE 대장균 및 Ni-NTA 아가로 오스 수지로 정제. 용출 된 단백질은 4 % -20 % 트리스 - 글리신 겔을 사용하여 SDS-PAGE 순도 분석 하였다. 심각한 열화 ZIF-EmGFP 융합 단백질의 잘림이 관찰되지 않았다. <img alt="그림 3" src="/files/ftp_upload/52814/52814fig3.jpg" /> 도 3 HeLa 세포 내로 전달 된 단백질을 ZIF는 매개. 두 손가락 ZIF-EmGFP 단백질의 양이 증가로 처리 된 HeLa 세포의 (A) 형광 강도. EmGFP 단백질로 처리 된 HeLa 세포 단독 미처리 세포와 완전히 중첩된다. (B) 2 μM o를 연속적으로 처리 된 HeLa 세포의 표준화 형광 강도F 두 손가락 ZIF-EmGFP 단백질. 형광 강도는 유동 세포 계측법으로 측정 하였다.

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

징크 핑거 도메인은 본질적으로 세포 투과성 및 포유 동물 세포 유형 내로 다양한 단백질 전달을 매개 할 수있다. 여기서, 단백질의 세포 내 전달을위한 아연 핑거 기술을 구현하기위한 상세한 단계별 프로토콜이 제시된다.

세포 투과성의 Cys를 사용하여 포유 동물 세포에 직접 단백질 배달<sub&gt; 2</sub&gt; - 그의<sub&gt; 2</sub&gt; 아연 손가락 도메인

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Nanyang Technological University

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K 조회수.  The Scripps Research Institute.  징크 핑거 도메인은 본질적으로 세포 투과성 및 포유 동물 세포 유형 내로 다양한 단백질 전달을 매개 할 수있다. 여기서, 단백질의 세포 내 전달을위한 아연 핑거 기술을 구현하기위한 상세한 단계별 프로토콜이 제시된다. 

비디오: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

포유 동물 세포를 Trypsinizing 및 Subculturing

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

공학 세포 투과 단백질

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZr 맞춤 아연 손가락 Nucleases와 게놈 편집 (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

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 ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC - 면역 정량 프로테오믹스를 사용하여 포유 동물 세포에있는 단백질 상호 작용 파트너의 식별

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

인간 배아 줄기 세포에 표적으로 아연 손가락 핵산 분해 효소 향상된 유전자

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...