우리는 DRS 감사합니다. 넉넉한 Hep293TT 세포를 제공하는 재조합 AAV3 및 AAV - EGFP의 plasmids, 각각 박사 게일 톰린슨 그들의 종류의 선물 R. 주드 Samulski과 샤오 샤오. 이 연구는 국립 보건원에서 공중 보건 서비스 권한을 부여 부여 R01 HL - 076901, R01 HL - 097088, 및 P01 DK - 058327 (프로젝트 1) (로)에 의해 부분적으로 지원되었다.

1. 재조합 Adeno - 관련 바이러스 혈청형 3 (rAAV3) 벡터의 포장 <ol><li> 웹사이트로 이동 : &quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;과 DNA의 양을 계산합니다. </li><li> FBS와 항생제없이 중간에 미리 혼합 plasmids pHelper (아데노 바이러스 도우미), pACG2c3 (AAV 도우미) 및 pdsAAV - CB - EGFP (rAAV 벡터). 총 볼륨 열 15cm 두 접시를 위해 8750 μL해야합니다. </li><li> 단계 1.2 DNA 솔루션으로 PEI의 직접 1,250 μL를 (0.1 % M / V, Polysciences, 고양이 # 23966, 산도 4.5) 추가합니다. 몇 초 동안 소용돌이 DNA - PEI 솔루션입니다. </li><li> 실온 (RT)에서 5 분 복잡한 형태로 품어 DNA - PEI 솔루션입니다. </li><li> 15cm 2 판 당 DNA - PEI 솔루션 1 ML을 추가합니다. </li><li> 신선한 25 ML 완벽한 매체로 대체, 4 시간 후 배지를 제거 10% FBS (C - DMEM). </li><li> 세븐티 이시간 이후 transfection, 긁어 세포와 250 ML 원뿔 원심 튜브에 부어. </li><li> 4 3,000 rpm으로 회전, ° 10 분 C. 뜨는을 하거라. </li><li> 다시 중지 RB TMS 버퍼 (50 MM 트리스 - HCL, 150 MM NaCl, 산도 8.0) 5 ML에있는 세포 펠릿. 새로운 15 ML 원뿔 관을 전송합니다. </li><li> 37 10 분 및 해동을위한 드라이 아이스 - 에탄올 욕조에 고정 ° C 10 분. 세 번 반복합니다. </li><li> 4.8 M MgCl 2 단계 중 1 μL 및 Benzonase 2 μL (25 U / μL, Novagen, 고양이 # 70664-3)을 추가합니다. 37 소용돌이와 부화 ° 40 분 C. </li><li> 40 분 ° 4, 4000 RPM에서 C를 스핀 다운 및 (lysate를 명확히) 표면에 뜨는를 수집합니다. </li><li> 15 %의 두 ML, 25 % 2 ML, 40 %의 1.5 ML 후 13 ML 빠른 시일 원심 분리기 튜브 (베크 고양이 # 342413), 아래로 위로부터 하중 iodixanol 기울기 (OptiPrep, 고양이 # 1114542)에서 60 % iodixanol 1.5 ML. iodixanol 그라디언트의 상단에 뜨는 (1.12 단계)를로드합니다. RB TMS 버퍼로 튜브를 입력합니다. </li><li> 16에서 1 시간 동안 75,000 rpm으로 원심 분리기 ° C 베크맨 90Ti의 로터를 사용합니다. </li><li> 40-60% 경계에 주사기를 삽입하여 40 % iodixanol 분율를 수집합니다. </li><li> 한 50 ML 원뿔 관에서 40 % iodixanol 분율을 전송하고 버퍼 40 ML까지하게 (20 MM 트리스, 15 MM NaCl, 산도 8.5). </li><li> HiTrap Q HP 열 (GE 헬스케어, 고양이 # 17-1154-01)를 사용하여 여과 장치 조립. 다음 버퍼로 컬럼을 씻어 : 버퍼의 25 ML (20 MM 트리스, 15 MM NaCl, 산도 8.5), 버퍼 B (10 MM 트리스, 500 MM NaCl, 산도 8.5) 25 ML, 버퍼의 50 ML; 버퍼에서 바이러스 샘플의 40 ML, 버퍼 50 ML (20 MM 트리스, 15 MM NaCl, 산도 8.5), 버퍼 C (20 MM 트리스, 175 MM NaCl, 산도 8.5) 20 ML; </li><li> 20 ML 아폴로 Concentrators (올비털 Biosciences)에 버퍼 C를 수집합니다. 4 3,000 rpm으로 원심 분리기, ° C 10 분. </li><li> 흐름 -을 통해을 취소하고 4, 3000 RPM 20 냉장 PBS의 ML과 원심 분리기를 추가 ° C, 10 분. </li><li> 흐름 -을 통해를 버리고 차가운 PBS 500 μL를 추가합니다. -80에서 작은 aliquots에 얼어 붙 어서 실리콘 - 대우 Eppendorf 튜브 및 저장하는 멤브레인 및 전송 ° C.에서 벡터를 씻어 </li></ol> 2. rAAV3 벡터 Titers의 결정 <ol><li> 얼음 Eppendorf 튜브에 바이러스를 정화 10 μL를 녹여. ddH 2 O. 40 μL 추가 </li><li> Benzonase의 0.2 μL (25 U / μL, Novagen, 고양이 # 70664)을 추가하고 37 품어 ° C를 1 시간. </li><li> 다른 Eppendorf 튜브에 플라스미드 표준 (0.2 NG / μL) 50 μL를 가져가라. </li><li> 65 각 튜브와 부화 ° C 30 분 100 MM NaOH 50 μL를 추가합니다. </li><li> 적어도 5 분 즉시 얼음 칠. </li><li> 바이오 점 여과지 (바이오 - 래드, 고양이 # 1620161)에 따라 전송 막 (Millipore, 고양이 # INYC00010)을 잘라 버릴거야. 20 분 세 여과지와 10X SSC의 막을 린스. </li><li> 슬롯 얼룩 SF 장치 (바이오 점, 고양이 # 170-6542)를 설정합니다. 진공 플라스크에 연결하고 슬롯 얼룩 장치에 10X SSC 100 μL를 추가합니다. </li><li> 단계 2.5의 샘플 각 20x SSC 100 μL와 배의 로딩 색소 1 μL를 추가합니다. </li><li> 슬롯 얼룩 장치에서 막에 각 시료 100 μL를로드합니다. </li><li> 샘플의 각으로 10X SSC의 또 다른 100 μL를 추가합니다. </li><li> 모든 예제는 세포막을 통해 될 때까지 단계 2.9와 2.10를 반복합니다. </li><li> 슬롯 얼룩 장치를 분해하여 세포막을 제거하십시오. 경감 링커 SL - 1000 자외선 Crosslinker으로 넣어요. &quot;에너지&quot;를 켭니다 &quot;최적 Crosslink&quot;(쇼 1200) 및 &quot;시작&quot;푸시. </li><li> 연어 정자의 종기 일 ML (SS) DNA (피셔, 고양이 # NC9753983) 5 분. 적어도 5 분 얼음 칠. </li><li> ddH 2 O, 15 ML의 27.5 ML을 혼합하여 사전 하이브 리다이 제이션 솔루션을 준비 20x SSC, 50x Denhardt의 솔루션 5 ML, 20 % SDS의 1.25 ML, PolyA의 0.25 ML과 단계 2.13에서 SS DNA 1 ML. </li><li> ° 하이브리드화 챔버에서 C 야간 65 덮인 유리 실린더와 부화에 사전 하이브 리다이 제이션 솔루션의 25 ML에 멤브레인을 일시 중지합니다. </li><li> ddH 2 O 10 μL의 50 NG DNA 템플릿을 희석. 100 삶아 ° C 이상에서 5 분 즉시 얼음에 5 분 및 진정하십시오. </li><li> 1 분에 대한 RT에서 3,000 rpm으로 원심 분리기. 10 MM 3 dNTPs 2 μL (dCTP를 부족), Hexanucleotide 믹스 2 μL (로체, ​​고양이 # 11277081001) Klenow 1 μL (로체, ​​고양이 # 11008404001)와 α -32 P - dCTP 5 μL를 추가합니다. 37 ° C 10 분 알을 품다. </li><li> 2 분 3,000 RPM에서 G - 50 열 (GE 헬스케어, 고양이 # 27-5330-01) 원심 분리기. 열에 단계 2.17에서 프로브를로드합니다. 2 분 3,000 rpm으로 원심 분리기와 흐름을 통해 수집합니다. </li><li> 방사능 백작. ° C 즉시 얼음에 5 분 및 진정에 대한 최소한 5 분 100에 프로브를 끓여. </li><li> 막과 사전 하이브 리다이 제이션 솔루션에 프로브를 (6x10 5 CPM / ML) 추가합니다. </li><li> 하이브리드화 챔버 65 ° C에서 밤새 품어. </li><li> 하이브 리다이 제이션 솔루션을 무시하고 15 분 RT에서 2X SSC 0.1 % SDS에서 막을 린스. </li><li> 솔루션을 버리고 ° C 30 분 65 0.1x SSC 0.1 % SDS에서 막을 린스. </li><li> 솔루션을 삭제하고 알티 간략에서 0.1x SSC에서 막을 린스. </li><li> 6 시간 -80 ° C에서 필름을 노출하고 필름을 개발합니다. 전형 결과는 그림 1에 표시됩니다. </li></ol> 3. rAAV3 벡터 - 중재 전달 및 인간 간암 세포에 Transgene 표현 <ol><li> 96 - 웰 플레이트의 각 우물에 씨앗 1x10 4 셀 (Huh7 또는 Hep293TT 중). 최소한 18 시간 동안 humidified CO 2 인큐베이터에서 부화. </li><li> 96 - 웰 플레이트에서 매체를 제거합니다. </li><li> FBS와 항생제없이 중간 50 μL로 두 번 세포를 씻고 제거합니다. </li><li> 5000 vgs / 휴대폰 FBS와 항생제와 rAAV 벡터 않고 중간의 50 μL를 추가합니다. </li><li> 4 시간 동안 CO 2 배양기에서 번호판을 품어. </li><li> 매체 폐기하십시오. 전체 매체 50 μL로 두 번 세포를 씻고 제거합니다. </li><li> 각 잘하는 C - DMEM 150 μL를 추가하고 72 시간 CO 2 배양기에서 접시를 부화하고, 형광 현미경을 사용하여 EGFP 식을 시각화 (DMI 4000B을, Leica 마이크로 시스템즈). </li><li> 바이러스에 감염된 세포의 세 웰스의 이미지는 ImageJ 분석 소프트웨어 (NIH, 베데스다, MD, 미국)에 의해 양적 분석하고 있습니다. </li><li> Transgene 표현은 영상 분야마다 녹색 형광의 총면적 (픽셀 2)로 평가됩니다. 전형 결과는 그림 2에 표시됩니다. </li><li> Transgene 표현도 유동세포계측법에 의해 결정하실 수 있습니다. </li></ol> 4. 대표 결과 : 위에서 설명한 프로토콜에 따라, 하나는 효율적으로 AAV의 혈청형 벡터를 생성할 수 있습니다. 전형적인 수확량은 500 μL ~ 10 11 vgs 포함 / 벡터 주식을 정화의 μL. 벡터 주식의 순도가와 양적 DNA 슬롯 blots에서 하이브 리다이 제이션 신호를 비교하여하고 Benzonase는 소화하지 않고 결정됩니다. 효율적으로 세포 라인 - hepatoblastoma (HB)와 간세포암 (HCC) - rAAV3 벡터 transduce 인간 간암을 정화. <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="그림 1" /> 그림 1. rAAV3 벡터의 titers을 결정하는 정량 DNA 슬롯 얼룩 분석. Benzonase (맨 윗줄)과 소화 바이러스 주식, 정화의 2 배 dilutions는 32 P - 라벨 EGFP 특정 DNA 프로브와 양적 DNA 슬롯 blots에서 분석했다. AAV3 벡터의 titer는 1 NG (중앙 행) 또는 멤브레인에 로드된 AAV - EGFP 플라스미드 기준 10 NG (하단 행)과 비교에 의해 결정되었다. 숫자는 DNA 사본과 일치합니다. <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="그림 2" /> 그림 2. 재조합 AAV 인간 간암 세포에 벡터 중재 transgene 표현. Hep293TT 세포, 최근에 설립된 인간 hepatoblastoma 전지 라인 4, 중 모의에 감염된 (왼쪽 패널)되었다, 또는 2 시간 동안 37 ° C에서 scAAV2 - EGFP 또는 scAAV3 - EGFP 벡터 중 5,000 vgs / 세포와 transduced. Transgene 표현 72 시간 이후 도입은 형광 현미경을 사용하여 시각되었습니다.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+PDH+deficiency+using+AAV8-siRNA+vector-mediated+knockdown+of+pyruvate+dehydrogenase+E1&#945;.">An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1&#945;.</a> Mol Genet &#38; Metabol. 101, 183-191 (2010).</li><li>Petrs-Silva, H., Dinculescu, A., Li, Q., Deng, W. T., Pang, J. J., Min, S. H., Chiodo, V., Neeley, A. W., Govindasamy, L., Bennett, A., Agbandje-McKenna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+properties+of+tyrosine-mutant+AAV2+vectors+in+the+mouse+retina.">Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina.</a> Mol Ther. , (2011).</li></ol>

관심 없음 충돌 선언하지 않습니다.

비 병원성 인간의 파르 보 바이러스, adeno - 관련 바이러스 (AAV)에 따라 재조합 벡터가 개발하고, 유전자 치료 임상 시험 5 여러 사용 현재되었습니다. 이전에는 10 개의 가장 일반적으로 사용되는 AAV의 serotypes의 일반적으로 AAV3 벡터의 전달 효율은 체외에서 생체내 1 두, 특히 낮은 것으로보고되었습니다. 그러나, 최근 관찰되는 AAV3로 유동세포계측법 2 양적 결정 매우 잘 벡터 transduce 인간 간암 세포 라인, 그 AAV3이에 바인딩 및 항목에 대한 공동 수용체 세포로 인간 hepatocyte 성장 인자 수용체 (hHGFR)를 활용 세포 3, 강하게 특히 일반적으로 인간의 간암 세포에 대한 AAV3의 선택 조직 tropism, 인간 간암 세포를 권장합니다. 그러나, 이후 AAV3 벡터는 또한 정상적인 인간 hepatocytes에게 효율적으로 2 transduce, 생체내에서 암 유전자 치료 응용 프로그램에서 자신의 잠재적인 사용에 부정적인 영향을 것입니다. 이 잠재적인 문제를 우회하는 한 가지 가능한 전략과 관련된 transcriptional 타겟팅 정상 hepatocytes에 의해 선택적으로 종양에 의해 생산되는 유전자 제품을 식별하여 암 세포하지만. 정상 hepatocytes은 일반적으로 매우 소량을 생산 이후 예를 들어, 이전의 연구 결과, α - 페토 프로테인 (AFP)의 혈청 수준이 존재, 진행 및 / 또는 간 암의 특정 유형의 reoccurrence를 추적하기위한 구체적인 표지로 사용하는 것으로 나타났습니다 이 단백질. 사실, 우리는 간암 세포에서 transgene 표현을 대상으로 AFP 프로 모터를 포함하는 AAV3 벡터를 사용한 있지만 정상적 hepatocytes 2, 연구의 현재 간암의 murine 이종 이식 모델에서이 접근 방식의 효능을 테스트하기 위해 진행되고 있습니다. 우리가 추가적인 연구에서 우리는 다양한 AAV의 혈청형 벡터의 전달 효율이 상당히 6-14 바이러스 capsids의 표면 노출 티로신 잔류의 사이트 이동 돌연변이 유발에 의해 증가된 수 있습니다 관찰했습니다. 6 7 표면 노출 tyrosines도 AAV3에 보존되어 있기 때문에, 우리는 이러한 잔류물의 사이트 감독 돌연변이 유발을 수행하고 티로신 - 돌연변이 AAV3 벡터의 전달 효율이 대폭 인간 간암 세포 (게시되지 않은 데이터)에 향상된 것을 관찰했습니다. 연구는 또한 인간 hepatoblastoma 및 간세포암에 대한 마우스 이종 이식 모델에서 티로신 - 돌연변이 AAV3 벡터의 안전성과 효능을 평가하기 위해 현재 진행되고 있으며, 성공적인 경우, 최적의 티로신 - 돌연변이 AAV3의 혈청형 벡터는 인간의 간을를 타겟팅하는 유용하다는 것을 증명하기 수 있습니다 잠재적인 유전자 치료에 대한 암.

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<td>DMEM</td>
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high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

비 병원성 인간 파르 보 바이러스에 따라 재조합 벡터는, adeno - 관련 바이러스 2 (AAV2)가 개발하고, 유전자 치료 임상 실험의 여러 사용 현재되었습니다. 최근 추가 AAV의 serotypes의 숫자도 다양한 크고 작은 동물 모델 1 조직 tropism 선택을 전시 게재된 보유하고있는, 고립되었습니다. 10 가장 일반적으로 사용되는 AAV의 serotypes의 AAV3는 체외에서뿐만 아니라 생체내의 세포와 조직을 transducing에 의해 지금까지 가장 효율적입니다. AAV3는 바인딩을위한 공동 수용체 세포로 인간 hepatocyte 성장 인자 수용체를 이용하기 때문에 매우 효과적으로 세포 라인 - hepatoblastoma (HB)와 간세포암 (HCC) - 그러나, 우리가 최근에 출판된 연구에, 우리는 AAV3 벡터 인간 간암 transduce 것을 문서화해야 이러한 세포 2,3에서 항목. 이 문서에서는, 우리는 기자의 유전자를 운반 재조합 벡터로 AAV3 인간 간암 세포의 고효율 전달을 달성하는 데 필요한 단계를 설명합니다. 치료 유전자를 운반 재조합 AAV3 벡터의 사용은 결국 인간의 간 암의 가능성 유전자 치료가 발생할 수 있습니다.

Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

이 문서에서는, 우리는 인간 간암 세포를 타겟팅하는 가장 효율적인 벡터로 adeno - 관련 바이러스 혈청형 3 (AAV3)의 신분증을 설명합니다.

재조합 Adeno - 관련 바이러스 혈청형 3 벡터에 의해 간암 세포의 고효율 전달

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

high-efficiency-transduction-liver-cancer-cells-recombinant-adeno

Research

JoVE Journal

Medicine

13.1K 조회수.  University of Florida. 이 문서에서는, 우리는 인간 간암 세포를 타겟팅하는 가장 효율적인 벡터로 adeno - 관련 바이러스 혈청형 3 (AAV3)의 신분증을 설명합니다.

비디오: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

연구

의학

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

분자 대장 암 세포의 3 차원 모델의 침윤성 종양 미세 환경의 프로파일 및<em&gt; 예 생체 내</em&gt; 섬유 아세포

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

도구로 아데노 관련 바이러스를 사용하여 질병 망막 장벽을 연구하는

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

재조합 아데노 - 관련 바이러스 성 벡터의 정위 주사를 통해 파킨슨 병에 대한 알파 - 시누 클레인 기반 쥐 모델 개발

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

에서 재조합 효소 중재 카세트 교환에 의해 재조합 인간 다 능성 줄기 세포의 신속하고 효율적인 생성<em&gt; AAVS1</em&gt; 로커스

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Adeno 관련 바이러스 중재 배달 심장 유전자 쥐에서 편집에 대 한 CRISPR의

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

작은 동물 모델에서 아데노 관련 바이러스 벡터의 결막하 투여

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

이중 요오딕사놀 밀도 구배 원심분리에 의한 AAV 분리

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

저자 스포트라이트: 전임상 애플리케이션을 위한 효율적인 아데노 관련 바이러스 분리

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.