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>

<ol>
	<li>Berg, A., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+domain+delivery+of+therapeutic+macromolecules.">Protein transduction domain delivery of therapeutic macromolecules.</a> Curr. Opin. Biotechnol. 22, 888-893 (2011).</li><li>Lindsay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-mediated+cell+delivery:+application+in+protein+target+validation.">Peptide-mediated cell delivery: application in protein target validation.</a> Curr. Opin. Pharmacol. 2, 587-594 (2002).</li><li>Luo, D., Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+DNA+delivery+systems.">Synthetic DNA delivery systems.</a> Nat. Biotechnol. 18, 33-37 (2000).</li><li>Guo, X., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+nonviral+vectors+for+gene+delivery.">Recent advances in nonviral vectors for gene delivery.</a> Acc. Chem. Res. 45, 971-979 (2012).</li><li>Thomas, C. E., Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat. Rev. Genet. 4, 346-358 (2003).</li><li>Frankel, A. D., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+uptake+of+the+tat+protein+from+human+immunodeficiency+virus.">Cellular uptake of the tat protein from human immunodeficiency virus.</a> Cell. 55, 1189-1193 (1988).</li><li>Elliott, G., O'Hare, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+trafficking+and+protein+delivery+by+a+herpesvirus+structural+protein.">Intercellular trafficking and protein delivery by a herpesvirus structural protein.</a> Cell. 88, 223-233 (1997).</li><li>Derossi, D., Joliot, A. H., Chassaing, G., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+helix+of+the+Antennapedia+homeodomain+translocates+through+biological+membranes.">The third helix of the Antennapedia homeodomain translocates through biological membranes.</a> J. Biol. Chem. 269, 10444-10450 (1994).</li><li>Smith, B. A., 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=Minimally+cationic+cell-permeable+miniature+proteins+via+alpha-helical+arginine+display.">Minimally cationic cell-permeable miniature proteins via alpha-helical arginine display.</a> J. Am. Chem. Soc. 130, 2948-2949 (2008).</li><li>Daniels, D. S., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+cell-permeable+miniature+proteins+based+on+a+minimal+cationic+PPII+motif.">Intrinsically cell-permeable miniature proteins based on a minimal cationic PPII motif.</a> J. Am. Chem. Soc. 129, 14578-14579 (2007).</li><li>Karagiannis, E. D., 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=Rational+design+of+a+biomimetic+cell+penetrating+peptide+library.">Rational design of a biomimetic cell penetrating peptide library.</a> ACS Nan. 7, 8616-8626 (2013).</li><li>Gao, S., Simon, M. J., Hue, C. D., Morrison, B., 3r, S., Banta, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unusual+cell+penetrating+peptide+identified+using+a+plasmid+display-based+functional+selection+platform.">An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform.</a> ACS Chem. Biol. 6, 484-491 (2011).</li><li>Fuchs, S. M., Raines, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+grafting+to+endow+cell+permeability.">Arginine grafting to endow cell permeability.</a> ACS Chem. Biol. 2, 167-170 (2007).</li><li>Cronican, J. 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=Potent+delivery+of+functional+proteins+into+mammalian+cells+in+vitro+and+in+vivo+using+a+supercharged+protein.">Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein.</a> ACS Chem. Biol. 5, 747-752 (2010).</li><li>Panyam, J., Labhasetwar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanoparticles+for+drug+and+gene+delivery+to+cells+and+tissue.">Biodegradable nanoparticles for drug and gene delivery to cells and tissue.</a> Adv. Drug Deliv. Rev. 55, 329-347 (2003).</li><li>Zelphati, O., 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=Intracellular+delivery+of+proteins+with+a+new+lipid-mediated+delivery+system.">Intracellular delivery of proteins with a new lipid-mediated delivery system.</a> J. Biol. Chem. 276, 35103-35110 (2001).</li><li>Kaczmarczyk, S. J., Sitaraman, K., Young, H. A., Hughes, S. H., Chatterjee, D. K. <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+engineered+virus-like+particles.">Protein delivery using engineered virus-like particles.</a> Proc. Natl. Acad. Sci. U. S. A. 108, 16998-17003 (2011).</li><li>Voelkel, C., 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=Protein+transduction+from+retroviral+Gag+precursors.">Protein transduction from retroviral Gag precursors.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 7805-7810 (2010).</li><li>Sinha, V. R., Trehan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+microspheres+for+protein+delivery.">Biodegradable microspheres for protein delivery.</a> J. Control Release. 90, 261-280 (2003).</li><li>Liu, J., Gaj, T., Patterson, J. T., 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=Cell-penetrating+peptide-mediated+delivery+of+TALEN+proteins+via+bioconjugation+for+genome+engineering.">Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering.</a> PLoS One. 9, e85755 (2014).</li><li>Ramakrishna, S., 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=Gene+disruption+by+cell-penetrating+peptide-mediated+delivery+of+Cas9+protein+and+guide+RNA.">Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.</a> Genome Res. 24, 1020-1027 (2014).</li><li>Fuchs, S. M., Raines, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyarginine+as+a+multifunctional+fusion+tag.">Polyarginine as a multifunctional fusion tag.</a> Protein Sci. 14, 1538-1544 (2005).</li><li>Mai, J. C., Shen, H., Watkins, S. C., Cheng, T., Robbins, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+protein+transduction+is+cell+type-dependent+and+is+enhanced+by+dextran+sulfate.">Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate.</a> J. Biol. Chem. 277, 30208-30218 (2002).</li><li>Al-Taei, S., 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=Intracellular+traffic+and+fate+of+protein+transduction+domains+HIV-1+TAT+peptide+and+octaarginine.+Implications+for+their+utilization+as+drug+delivery+vectors.">Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors.</a> Bioconjug. Chem. 17, 90-100 (2006).</li><li>Jones, S. W., 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=Characterisation+of+cell-penetrating+peptide-mediated+peptide+delivery.">Characterisation of cell-penetrating peptide-mediated peptide delivery.</a> Br. J. Pharmacol. 145, 1093-1102 (2005).</li><li>Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., Gregory, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+with+engineered+zinc+finger+nucleases.">Genome editing with engineered zinc finger nucleases.</a> Nat. Rev. Genet. 11, 636-646 (2010).</li><li>Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+with+zinc-finger+nucleases.">Genome engineering with zinc-finger nucleases.</a> Genetics. 188, 773-782 (2011).</li><li>Guo, J., 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=Directed+evolution+of+an+enhanced+and+highly+efficient+FokI+cleavage+domain+for+zinc+finger+nucleases.">Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.</a> J. Mol. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+and+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.</a> Trends Biotechnol. 31, 397-405 (2013).</li><li>Perez-Pinera, P., Ousterout, D. G., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+targeted+genome+editing.">Advances in targeted genome editing.</a> Curr. Opin. Chem. Biol. 16, 268-277 (2012).</li><li>Cronican, J. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Methods Mol. Biol. 91, 1-24 (1998).</li><li>Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., 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=Regulation+of+endogenous+human+gene+expression+by+ligand-inducible+TALE+transcription+factors.">Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.</a> ACS Synth. Biol. 3, 723-730 (2014).</li><li>Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., Horton, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

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...

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 없?...

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

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

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 ...

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 ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

포유 동물 세포에서 초기 DNA에 바인딩 단백질의 시험 BrdU의 칩 슬롯 서양 기술을 사용하여

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...