저자는이 원고에 대한 데이터를 기여 마이클 추, 빙빙 다이, 그리고 리앙 샤오를 인정하고 싶습니다. 우리는 또한이 연구에 사용 된 시약의 관대 선물 닥터 존 그레이을 인정합니다. PB는 국립 암 센터에서 박사 친교에 의해 지원됩니다. 이 연구는 건강의 국립 연구소 (R01AI68978, P01CA132681 및 RCA170820A), 빌과 멜린다 게이츠 재단, 번역 의학 합동 센터에서 번역 가속 그랜트와 캘리포니아 HIV / AIDS에서 부여에서 부여에서 교부금에 의해 지원되었다 연구 프로그램.

DC-LV 안정적인 생산 세포는 필요한 렌티 바이러스 구성 요소 gagpol, 회전 및 TET 오프 시스템을 포함 GPR 포장 세포주 11에 따라 구성됩니다. 첫째, 레트로 바이러스 전달은 TET 의존 SVGmu 당 단백질을 인코딩 GPRS 포장 세포주를 생성하는 데 사용됩니다. 그런 다음, concatemer 배열 형질이 같은 GFP와 같은 렌티 바이러스 벡터 유전자와 GPRS 세포주를 형질하는 데 사용됩니다. LV-MGFP로 지정이 안정 생산 세포주는, GFP에 대한 DC-LV 백신 생산 능력에 대한 in vitro와 in vivo에서 테스트 할 수 있습니다. 1. Tet의 의존 SVGmu 세포 라인을 생성 플라스미드 PRX-SVGmu는 DC-특정 당 단백질 SVGmu 하류 레트로 바이러스 플라스미드 pRetroX-Tet의 오프 1 (그림 1C)의 TTA-고급 발기인 복제되는 구조이다. D10의 문화 293T 세포 (Dulbecco의 수정 이글의 중간에10 % 소 태아 혈청, 2 mM의 L-글루타민). 독시사이클린 (1 NG / ML) 및 puromycin (2 ㎍ / ㎖)와 D10의 문화 GPR 포장 세포주. 모든 세포는 5 % CO 2 가습 37 ° C 배양기에서 배양된다. <ol><li> 16-18 시간 전에 일시적으로 형질 전환, 플레이트 2 × 10 세포를 형질 전환시 90 % confluency에 접근하는 등 6 cm 조직 문화 접시에있는 D10의 4 ML 6 293T 세포. </li><li> 레트로 바이러스 플라스미드의 형질 : 혼합 100 μL 1.25 M 염화칼슘 용액, 멸균 밀리 Q 물과 같은 플라스미드 : 5 μg PRX-SVGmu, 2.5 μg pGag 폴, 2.5 μg pVSV-G. 최종 부피가 500 μL입니다. 5 ML의 폴리스티렌 왕복 아래 튜브에 500 μL 2X HBS를 (50 MM의 HEPES, 10 mM의 KCl을 12 mM의 포도당, 280 mM의 NaCl을, 2 HPO 4 * 7H 2 O, pH가 7.05 나 1.5 밀리미터)를 추가합니다. 유리 파스퇴르 피펫으로 적극적으로 버퍼를 버블 링하는 동안 2X HBS 버퍼에 플라스미드 혼합물 적하를 추가합니다. 마일을 추가 한 후xture는 또 다른 30 ​​초 버블 링 계속합니다. 그런 다음, 6 cm 배양 접시에있는 293T 세포에 전체 혼합물을 추가하고, 37 품어 ° C. </li><li> 4 시간 형질 전환 한 후, 조심스럽게 4 ML 예열 D10과 문화 접시에있는 매체를 교체하십시오. </li><li> 감염 스핀 : 형질 전환 후 48 시간을, 수확 SVGmu - 인코딩 0.45 μm의 필터를 통해 상층 액을 통과하여 상층 액에서 레트로 바이러스 입자를. / 물론 2 × 10 4 세포의 24 잘 접시에 판 GPR 포장 세포. 1,050 XG에 90 분, 25 ° C.에 대한 접시에 GPR 포장 셀 (2 ㎖ /도)와 원심 분리기 세포 상층 액을 여과 추가 (2 ㎖ / 잘) 스핀 감염 후 독시사이클린 (1 NG / ML) 신선한 D10에 매체를 변경합니다. </li><li> 포장 세포가 독시사이클린 (1 NG / ML) 및 puromycin (2 NG / ML)와 D10의 형질 72 시간 후 형질은 문화를 확장합니다. </li><li> 48 시간 동안 독시사이클린없이 SVGmu, 문화, 세포의 발현을 확인합니다. 표면 EX를 측정안티 Sindbis 혈청을 사용하여 유동 세포 계측법에 SVGmu의 컴프레션. 이러한 세포는 GPRS 포장 세포주로 지정되어 있습니다. </li></ol> 2. Concatemer 배열 형질에 의해 DC-LV 생산자 세포를 생성 렌티 바이러스 전송 플라스미드 TL20-GFP는 자체 운영을 중지 렌티 바이러스 전달 벡터 플라스미드 독스 - 조절 가능한 바이러스 성 RNA 게놈 발현 시스템 7 TET 사업자 (그림 1C) 11와 사이토 메갈로 바이러스 (CMV) 증강의 교체와 pCL20c-MSCV-GFP를 기반으로 , 12,15. 플라스미드 PGK-BLE은 약한 PGK 프로 2에 의해 구동 블레오 마이신 내성 (BLE) 카세트 테이프입니다. <ol><li> 50에서 제한 효소 SfiI와 다이제스트 20 μg 플라스미드 TL20-GFP ° C. 37 PflMI와 다이제스트 20 μg 플라스미드 PGK-BLE ° C. </li><li> 아가로 오스 겔 전기 영동하여 DNA 조각을 (PGK-BLE 카세트 1011 BP와 벡터이다 TL20-GFP는 6861 bp의 경우) 정화. </li><li> TL20-GFP 벡터 및 PGK -을 결찰25:1의 몰비 (NEB T4 DNA ligase를)에서 BLE 카세트입니다. 실온에서 밤새 품어. </li><li> 하룻밤 결찰 후 결찰 혼합물 (Qiagen의 DNeasy의 혈액 및 조직 키트)에서 DNA를 정화. 순화 한 DNA의 양을 확인 5 μg 정도입니다. </li><li> 형질 전 16-18 시간, 자랄는 형질 전환시 약 90 %가 될 것 같은 6 cm 조직 문화 접시에 접시에 GPRS 포장 세포. </li><li> 6 cm 조직 문화 접시에 GPRS 포장 세포로 정제 concatemeric DNA 5 μg을 형질하는 단계 1.2에서 설명한 인산 칼슘의 transfection 방법을 사용합니다. 4 시간 형질 전환 한 후, 신중하게 매체를 제거하고 독시 싸이클린 (1 NG / ML) 4 ML 예열 D10로 교체합니다. </li><li> 48 시간 형질 전환 한 후, 독시사이클린 (1 NG / ML) 및 puromycin (2 ㎍ / ㎖)를 D10에 매체를 변경합니다. 문화 매체 zeocin을 (50 ㎍ / ㎖) 첨가하여 형질 전환 클론을 선택합니다. 세포 coloni까지 약 2 주 동안 문화 세포ES는 요리의 하단에 볼 수 있습니다. </li><li> 요리의 하단에있는 세포 식민지 레이블을 지정합니다. 24 잘 조직 배양 접시를 취할 수 우물과 각 well에 2 ㎖ D10 포함 zeocin (50 ㎍ / ㎖), 독시사이클린 (1 NG / ML) 및 puromycin (2 ㎍ / ㎖)을 추가 할 수 있습니다. 형질 세포의 매체를 대기음, 그 분 미만의 식민지의 각각에 트립신 한 방울을 추가합니다. 그런 다음, 같은 식민지에 D10의 하나 또는 그 이상의 방울을 추가합니다. 피펫 하나의 식민지 하나를 선택하고 24 잘 조직 배양 플레이트 별도의 우물에 그들을 전송합니다. </li><li> 문화와 바이러스 생산 능력 평가를위한 D10 포함 zeocin (50 ㎍ / ㎖), 독시사이클린 (1 NG / ML) 및 puromycin (2 ㎍ / ml)에있는 모든 세포 클론을 확장합니다. </li></ol> 3. 각 세포 클론의 바이러스 생산을 평가 <ol><li> 독시사이클린없이 그 자랄 같은 D10에서 6 cm 조직 문화 요리에 4X10 6 세포 주변의 프로듀서 세포와 플레이트를 Trypsinize90 %를 초과합니다. 매일 신선한 예열 D10와 매체를 교체하고 DOX를 제거한 후 역가 분석 72 시간 동안 매체를 수집합니다. </li><li> 0.45 μm의 필터 매체를 필터링하여 바이러스 상층 액을 수확. </li><li> 플레이트 대상 세포 × 10 4 세포 / 물론 1, 96 - 웰 배양 접시에서 음성 대조군으로 DC-SIGN 수용체 (예 : 파생 293T.hDC 서명 또는 마우스 골수 수지상 세포 16)와 293T를 표현. 우물 (물론 100 μL /)로 바이러스 상층 액의 2 배 연속 희석을 추가합니다. 보통 6-8 희석는 GFP 양성 세포가 추가 된 바이러스의 양에 선형 관계에있는 형질하는 범위에 도달하기에 충분합니다. </li><li> 세포를 원심 분리하고 단계 1.4에서 설명한 매체를 교체하십시오. BMDCs 10 % FBS와 GM-CSF (1시 20분 J558L 에어컨 매체)를 RPMI 배지에서 배양되어야한다. </li><li> 유동 세포 계측법 4-6일 후 전달하여 세포를 형질에서 GFP 발현을 측정한다. 렌티 바이러스 vectoGFP 양성 세포와 벡터 금액의 비율이 선형 관계에있을 때 R 역가가 벡터 희석 범위에서 계산됩니다. 이후의 응용 프로그램에 대한 높은 바이러스 생산 세포 클론을 선택합니다. </li></ol> 4. 렌티 바이러스 벡터를 생성하고 집중 <ol><li> 문화 15 cm 조직 문화 요리 생산자 세포주 (LV-MGFP). </li><li> 독시사이클린없이 신선한 D10에서 자랄 때 90 % 이상에서 15 cm 조직 문화 요리에 생산자 세포와 플레이트 세포를 Trypsinize. 매일 신선한 예열 D10과 매체를 변경합니다. </li><li> 피크 바이러스 생산 (같은 사용자에 의해 결정)시, 바이러스 상층 액을 수확하고 0.45 μm의 필터를 사용하여이를 필터링합니다. 90 분 동안 50,000 XG에 파라 필름 (parafilm) 및 원심 분리기, 4 ° C로 밀봉 두꺼운 벽 32.5 ML의 원심 분리기 튜브로 필터링 상층 액을로드합니다. 철저하게 50 μL 또는 응용 프로그램에 따라 PBS 또는 HBSS의 적절한 볼륨 펠렛을 resuspend을. &lt;/ 리&gt; </ol> 5. 생체 내 마우스를 면역 항원 특정 면역 반응을 분석 <ol><li> 4 절에 설명 된대로 높은 titer의 바이러스를 생산하고 있습니다. </li><li> 벡터 서스펜션 (발바닥 당 25 μL)와 노상 루트를 통해 BALB / C 마우스 (구, 여성 6-8주)을 주입. </li><li> 이주 예방 접종 후 비장과 수확 비장 세포를 분리. GFP 에피토프 펩티드와 GFP 특정 CD8의 존재 + 세포 내 사이토 카인 염색법을 사용하여 T 세포를 분석 문화 비장은 앞에서 17 (그림 3B) 설명했다. </li></ol>

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M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449, 419-426 (2007).</li><li>Yang, L., 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=Engineered+lentivector+targeting+of+dendritic+cells+for+in+vivo+immunization.">Engineered lentivector targeting of dendritic cells for in vivo immunization.</a> Nature Biotechnology. 26, 326-334 (2008).</li><li>Hanawa, H., 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=Efficient+gene+transfer+into+rhesus+repopulating+hematopoietic+stem+cells+using+a+simian+immunodeficiency+virus-based+lentiviral+vector+system.">Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.</a> Blood. 103, 4062-4069 (2004).</li><li>Yang, L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+provision+of+antigen-specific+T+cell+immunity+by+programming+hematopoietic+stem+cells.">Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 4518-4523 (2005).</li><li>Dai, B., 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=HIV-1+Gag-specific+immunity+induced+by+a+lentivector-based+vaccine+directed+to+dendritic+cells.">HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 20382-20387 (2009).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J. Vis. Exp. (63), e4031 (2012).</li><li>Kochan, G., Escors, D., Stephenson, H., Breckpot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Grade+Lentiviral+Vectors.">Clinical Grade Lentiviral Vectors.</a> Lentiviral Vectors and Gene Therapy. , 69-85 (2012).</li><li>Loew, R., 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+new+PG13-based+packaging+cell+line+for+stable+production+of+clinical-grade+self-inactivating+gamma-retroviral+vectors+using+targeted+integration.">A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.</a> Gene Ther. 17, 272-280 (2010).</li><li>Gaspar, H. B., 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+therapy+of+X-linked+severe+combined+immunodeficiency+by+use+of+a+pseudotyped+gammaretroviral+vector.">Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.</a> Lancet. 364, 2181-2187 (2004).</li><li>Cornetta, K., 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=Replication-competent+lentivirus+analysis+of+clinical+grade+vector+products.">Replication-competent lentivirus analysis of clinical grade vector products.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 19, 557-566 (2011).</li><li>Stewart, H. 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+stable+producer+cell+line+for+the+manufacture+of+a+lentiviral+vector+for+gene+therapy+of+Parkinson's+disease.">A stable producer cell line for the manufacture of a lentiviral vector for gene therapy of Parkinson's disease.</a> Human Gene Therapy. 22, 357-369 (2011).</li><li>Coroadinha, A. 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=Retrovirus+producer+cell+line+metabolism:+implications+on+viral+productivity.">Retrovirus producer cell line metabolism: implications on viral productivity.</a> Appl. Microbiol. Biotechnol. 72, 1125-1135 (2006).</li></ol>

<p class="jove_content"&gt; 저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.</p

여기, 우리는 안정적으로 규제 시스템의 전원 TET 아래의 모든 렌티 바이러스 구성 요소와 형질 293T 세포를 사용하여 렌티 바이러스 벡터의 대량 생산을위한 방법을 설명했다. 지금까지, 렌티 바이러스 벡터를 생산하는 대부분의 프로토콜 (예를 들어, 18 참조) 표준 인산 칼슘 일시적인 형질 전환에 의존하고 있습니다. 이 방법은 임상 척도에 성공했습니다,하지만 안정적인 세포 라인에서 ?...

많은 벡터 시스템은 유전자 전달을 위해 개발되었습니다. 렌티 바이러스에 기반 벡터는 가장 일반적으로 공부 바이러스 시스템 사이에 있었다. 그들이 효율적으로 세포 1 분할 및 비 나누어 모두 형질 도입으로 인해 호스트 게놈에 통합 장기적인 표현을 달성, 대부분의 인간 집단이 낮은 천연 안티 - 벡터 내성을 전시하고, 대한 낮은 가능성을 가질 수 있기 때문에 이러한 벡터 유익하다 insertional 돌연변이 3,4에서 유전 독성. 렌티 바이러스의 생산은 항상 안전에 대한 우려로 채색되어 있습니다. 렌티 바이러스 벡터는 일반적으로 HIV-1, AIDS의 원인균 에이전트에서 파생됩니다. lentivector의 개별 구성 요소 (전송, 봉투, 포장 플라스미드)의 일시적인 형질 실험실 설정에서 유전 물질을 전달하는 일반적이고 유연한 방법입니다. 그러나 임상 응용을위한 확장까지 과도의 transfections는 복잡하고 엄마입니다y를 복제 능력 렌티 5,6의 발전으로 이어질. 이러한 장애물을 극복하기 위해, 몇몇 안정 포장 및 생산 세포주는 6-11 개발되었다. 이 라인 중 하나 GPR 포장 라인 (11)은 테트라 사이클린에 의해 규제되는 매력적인 장점이 있습니다. 본 논문에서, 우리는 구체적으로 돌기 세포 (DC-정맥 주사) 12를 대상으로하는 자체 운영을 중지시키지 렌티 바이러스 벡터를 생산하는이 시스템을 적용하는 방법을 보여줍니다. 돌기 세포 (DCS)는 면역 시스템의 가장 강력한 항원 제시 세포입니다. 그들이 직접 프로그램을 시작하고, 종양 특이 면역 반응하기 13 규제 때문에 그들은 암 백신 개발에 큰 관심의 대상이되고있다. DC의 펩타이드 또는 DNA 백신보다 더 강력한 항암 면역 반응을 유도 할 수있는 잠재력을 포함하는 예방 접종 프로토콜을 통합. 최근에, 우리는 렌티 바이러스 벡터를 개발 한 specifically를 수정 sindbis 바이러스 당 단백질, SVGmu 14을 통해 수지상 세포를 대상으로합니다. 그들은 돌기 세포에 높은 특이성을 보여 비특이적, VSVg-pseudotyped 벡터보다 강한 면역 반응을 생성하는 이러한 벡터는 고유합니다. 여기, 우리는이 DC-타겟 렌티 바이러스 벡터의 대량 생산을위한 방법을보고합니다. 우리는이 DC-LV를이 수지상 세포를 감염하고 강력한 CD8 + T 세포의 면역 반응을 생성 할 수 보여줍니다. 동물과 관련된 모든 절차는 USC Intitutional 동물 관리 및 사용위원회의 승인에 따라 인도적으로 수행되었다. 생체 및 임상 실험을 수행하기 위해서는, 그것은 높은 역가에 바이러스를 생성 할 수 세포 라인을 만드는 것이 중요합니다. 설명대로 전달과 형질 전환 단계를 수행하면 이러한 복제를 생성하는 기회를 극대화하는 것입니다.

<table><tbody><tr><td>DMEM</td><td>Mediatech Inc.</td><td>10-013-CV</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>Glutamine</td><td>Mediatech Inc.</td><td>25-005-Cl</td><td></td></tr><tr><td>Doxycycline</td><td>Clontech Laboratories, Inc.</td><td>631311</td><td></td></tr><tr><td>Zeocin</td><td>Invitrogen</td><td>R250-01</td><td>Toxic</td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit</td><td>Qiagen</td><td>69506</td><td></td></tr></tbody></table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

렌티 바이러스 벡터 (정맥 주사)는 세포의 여러 유형에 유전 물질을 전달하는 강력한 수단입니다. 정맥 주사의 대량 생산이 HIV-1 유래 벡터와 관련된 안전 문제 때문에하는 것은 도전이다. 본 논문에서, 우리는 자기 불활 정맥 주사의 높은 역가를 생산하는 방법을보고합니다. 우리는 retrovirally 돌기 세포 특정 당 단백질, SVGmu 포함한 모든 바이러스 구성 요소를 표현할 수있는 세포주, GPRS를, 생성하는 TET 오프 안정적인 생산 세포주 GPR를​​ 형질 도입. 다음, 우리는 LV 전송 플라스미드 인코딩에게 선택 마커와 함께 리포터 유전자 GFP를 형질을 concatemeric DNA의 형질 전환을 사용합니다. 결과 클론 중 일부는 우리가 일시적인 형질로 얻을 수있는 것보다 10 배 더 큰 역가에 LV를 생성 할 수 있습니다. 또한, 이러한 바이러스를 효율적으로 체외에서 수지상 세포를 형질 도입하고 기자 항원에 강한 T 세포 면역 반응을 생성합니다. 이 방법은 좋은 옵션을 구경 할 수 있습니다R 암 또는 감염성 질환의 임상 연구에 대한 강력한 LV-기반의 백신을 생산.

이 방법에서 설명하는 안정적인 세포 라인은 특히 돌기 세포를 대상으로하는 렌티 바이러스 벡터의 대량 생성 할 수 있습니다. 그림 1B에서 보는 바와 같이, 각각의 클론의 분리 품질 12 변화의 안정적인 세포 라인을 굴복. 테스트 26 클론 중 8 일시적인 형질 전환에 의해 생산 SVG-pseudotyped 렌티 바이러스 벡터에 대한 일반적인 벤치 마크 ML (TU / ML), 당보다 큰 10 6 전달 단?...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

여기, 우리는 레트로 바이러스 전달 및 테트라 사이클린의 부재 렌티 바이러스 벡터 (LV)의 구성 요소를 표현할 수있는 세포 라인을 만들 수 concatemeric 형질을 사용합니다. 이 LV는 GFP를 인코딩 및 수지상 세포 수용체 특정 당 단백질, SVGmu로 pseudotyped 있습니다.

항생 물질 규제 세포주 특히 수지상 세포를 대상으로 높은 titer의 렌티 바이러스 벡터를 생성

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.5K 조회수.  University of Southern California, Los Angeles. 여기, 우리는 레트로 바이러스 전달 및 테트라 사이클린의 부재 렌티 바이러스 벡터 (LV)의 구성 요소를 표현할 수있는 세포 라인을 만들 수 concatemeric 형질을 사용합니다. 이 LV는 GFP를 인코딩 및 수지상 세포 수용체 특정 당 단백질, SVGmu로 pseudotyped 있습니다.

비디오: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

연구

면역학 및 감염병학

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus 특정 T 세포 생성 Allogeneic 조혈 줄기 세포 이식 후 바이러스 감염을 예방 / 치료

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

세포 성숙없이 돌기 세포의 효율적인 Transfection을 위해 최적화된 프로토콜

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

인간으로 Epigenome 편집 도구의 효율적인 배달 lentiviral 벡터 플랫폼 유도 만능 줄기 세포 유래 질병 모델

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

유전학

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

중추 신경계에서 Transducing 전지 Lentiviral 벡터 제작

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

신경과학

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

테트라싸이클린-inducible (Tet-에서) shRNA 또는 cDNA 표현과 안정적인 인간 세포 라인의 생성

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

생물학

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

유전자 변형 T 세포 생성을 위한 GMP 준수 절차

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

생체공학

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate na&#239;ve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.

대장포에이성 줄기및 전구세포주에서 짧은 헤어핀 RNA 중재 유전자 녹다운에 의한 수지상 세포 개발 연구

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided ...

<meta charset="utf8"></head><body>In Vivo 유전자 조절을 위한 Lentiviral 벡터 준비

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>저자 스포트라이트: Exploring Macrophage Immunometabolism Through Lentiviral Vector-Mediated Gene Manipulation (렌티바이러스 벡터 매개 유전자 조작을 통한 대식세포 면역대사 탐색)

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

생쥐의 In Vivo 에서 복막강 조직 거주 대식세포의 효율적인 유전자 및 MicroRNA 조절을 위한 렌티바이러스 벡터 준비

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...