Name: retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation
Uploaded: 2014-10-20T15:00:00.000+00:00
Duration: 11 min 40 s
Description: Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1 배아 바디 (EB) 형성 <ol><li> 마우스 배아 섬유 아 세포 (MEFs)에 유지 된 ESCS을 젤라틴 - 적응. 통로 ESCS MEFs을 제거하기 위해 0.1 % 젤라틴으로 코팅 여섯 잘 조직 배양 접시에 세 번. <ol><li> (표 1) 미분화 세포를 유지하기 위해 함께 LIF ESC 유지 매체를 세포 성장. 확인 세포가 결코 80 %의 합류를 초과하지합니다. 분화 세포 (MEFs에서 제거한 후 10 이하 통로) 낮은 통로를 사용합니다. </li></ol></li><li> ESCS는 EB의 분화 배양되기 전에 날, 통로 세포는 그래서 그들은 약 50-70% 합류 다음 날이 될 것입니다. 세포의 다 능성을 유지하는 ESC 유지 배지에서 세포 배양을 계속한다. </li><li> 6 웰 플레이트 잘 당 : 분화, 흡인 ESC 유지 보수 매체와의 일에 1 ㎖의 인산염 완충 생리 식염수 (137 mM의 염화나트륨, 2.7 밀리미터의 KCl, 11.9 mM의 인산염 완충액 pH 7.4의 PBS)으로 씻는다. <ol><li> 0.25 %의 tryps의 200 μl를 추가/ 각 EDTA 잘에서 3 분 동안 37 ° C에서 배양한다. </li><li> 차별화 매체의 800 μL와 트립신 / EDTA를 비활성화합니다. 피펫 위아래 P200 팁 2 ㎖의 혈청 학적 피펫에서 휴식 - 최대 세포를 단일 세포 현탁액으로. 미디어 피펫 팁 인터페이스 사이에 이동되지 않도록 P200 팁 끝에 딱 맞는 있는지 확인합니다. </li><li> 피펫은 위아래로 약 5 ~ 10 배. 피펫 팅하는 동안 거품을 만들 않도록주의하십시오. </li></ol></li><li> 15 ML 원뿔 튜브에 세포를 전송합니다. PBS 10 ML에 볼륨을 가져와. 실온에서 5 분 동안 315 XG에 원심 분리기. </li><li> 상층 액을 제거합니다. LIF를 포함 남은 용지를 제거하는 5 ㎖의 PBS로 두 번 세척 할 것. 각 세척 단계와 1.4에서와 같이 원심 분리기. <ol><li> 분화 매체 (표 2) 2 ㎖의 최종 세포 펠렛을 재현 탁. </li></ol></li><li> 혈구 또는 셀 카운터 세포를 세어 10cm 페트리 접시에 6,000-10,000 세포 / ml를 판(비 조직 배양 치료). <ol><li> 각 ESC 라인에 대한 경험적으로 정확한 세포 / ml를 결정합니다. 일반적으로 세균 작업 또는 낮은 부착 판에 사용되는 멸균 페트리 접시를 사용합니다. 주 : 최상의 차별화 결과, 사채가 판에 연결하지 않고도 정지에 남아있는 구체를 형성해야한다. 최소한의 준수와 사람을 찾기 위해 판의 여러 브랜드를 테스트합니다. </li><li> 5 % CO 2와 37 ° C 조직 문화 인큐베이터에서 차별화 ESCS을 품어. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> 시약 </td><td> 주식 </td><td> 최종 농도 </td><td> 볼륨 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> 1 배 </td><td></td><td> 410 ml의 </td><td> S<html> IGMA / 알드리치 </td><td> D5796 </td></tr><tr><td> FBS (ES 선별) </td><td> 백퍼센트 </td><td> 15 % </td><td> 75 ML </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> 비 필수 아미노산 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 11140 </td></tr><tr><td> L-글루타민 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 35050 </td></tr><tr><td> Penncillin / 스트렙토 마이신 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 15070 </td></tr><tr><td> β-머 캅토 에탄올 </td><td> 14.3 M </td><td> 114 μM </td><td> 4 μL </td><td> 시그마 / 알드리치 </td><td> M3148 </td></tr><tr><td> 백혈병 억제 인자 (LIF) </td><td> 10 7 단위 / ㎖ </td><td> 1,000 개 / ㎖ </td><td> 50 μL </td><td> 밀리 포아 </td><td> ESG1107 </td></tr></tbody></table>&quot;ontent&gt; 표 1 : ESC 유지 미디어. <table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> 시약 </td><td> 주식 </td><td> 최종 농도 </td><td> 볼륨 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> 1 배 </td><td></td><td> 410 ml의 </td><td> 시그마 / 알드리치 </td><td> D5796 </td></tr><tr><td> FBS (사용자가 최적의 차별화에 대한 검사) </td><td> 백퍼센트 </td><td> 15 % </td><td> 75 ML </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> 비 필수 아미노산 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 11140 </td></tr><tr><td> L-글루타민 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> 생활 TECHNOLOGIES </td><td> 35050 </td></tr><tr><td> Penncillin / 스트렙토 마이신 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 15070 </td></tr><tr><td> β-머 캅토 에탄올 </td><td> 14.3 M </td><td> 114 μM </td><td> 4 μL </td><td> 시그마 / 알드리치 </td><td> M3148 </td></tr></tbody></table> 표 2 : 차별화 미디어. 바이러스 감염에 대한 2 준비 EB 세포 <ol><li> 개발의 원하는 단계에서 (일 2 일 3 사이), 15 ML 원뿔 관에 10cm 페트리 접시에서 교환 사채를 전송합니다. 5 ㎖의 PBS로 접시를 씻고 원뿔 튜브에 세척을 추가합니다. </li><li> 5 분 동안 실온에서 315 XG에 사채 펠렛. 5 ㎖의 PBS와 EB 세포를 씻으십시오. </li><li> 해동 세포 분리 솔루션 (포함하는 단백질 분해 및 collagenolytic 효소) 1 ㎖를 추가하고 OCC 30 분 동안 37 ° C의 물을 욕조에 튜브 (또는 창업)을 배치(플릭 또는 빛 소용돌이로 교반부터) asional 교반. </li><li> 차별화 미디어의 한 ML을 추가합니다. 상하 피펫 P200 피펫 팁 2ml를 피펫으로. 30 ~ 60 분 동안 다시 37 ° C의 물을 욕조에 튜브를 놓습니다. </li><li> 5 분 동안 315 XG에서 세포를 스핀 다운 및 PBS로 세포를 씻어. </li></ol> 3 바이러스 감염 <ol><li> 분화 매체의 1.5 ml의 세포를 재현 탁. 6 잘 아닌 조직 문화 처리 플레이트에 세포 현탁액을 전송합니다. </li><li> 미리의 표준 기술에 의해 레트로 바이러스를 준비합니다. 바이러스 역가에 따라 세포에 바이러스 뜨는 5-100 μl를 추가합니다. 이 사채의 후속 개혁을 억제로 감염을 향상시키기 위해 폴리 브렌를 추가하지 마십시오. </li><li> 실온에서 90 분 동안 2,120 XG에서 원심 분리하여 바이러스와 세포를 Spinoculate. </li></ol> 4 매달려 방울에 바이러스 성 감염 EB 세포 차별화 <ol><li> 난의 각 웰에 차별화 미디어의 두 ML을 추가nfected EB 세포 현탁액. </li><li> 피펫 multipipettors에 대한 멸균 시약 저수지에 감염된 EB 세포 현탁액의 3.5 ML. 팔 20 μL의 15 ~ 20 행 15cm 페트리 접시의 반전 뚜껑에 방울을 피펫하는 multipipettor를 사용합니다. </li><li> 페트리 접시의 아래쪽에 멸균 PBS 또는 물 10ml를 추가합니다. 그런 다음 페트리 접시에 방울과 장소 뚜껑을 반전. 방울 챔버 가습 유지하기 위해 거꾸로 PBS 이하 물 뚜껑 매달려되어 있는지 확인합니다. </li><li> 5.0 %의 CO 2와 37 ° C 조직 문화 인큐베이터에서 접시를 놓습니다. </li><li> 이일 후, 차별화 미디어의 4.5 ml의 뚜껑을 세척 반전에 의해 삭제 교수형에 형성 사채를 수집합니다. <ol><li> 새 10cm가 아닌 조직 배양 페트리 접시로 전송 미디어 및 사채. 차별화 미디어와 10cm 플레이트로 전송의 3 ㎖로 한 번 뚜껑을 세척 할 것. </li></ol></li><li> 세포 계측법 (8)의 총 후 흐름에 의한 분석을위한 수확 세포차별화 미디어 (-LIF)에 대한 세포의 초기 전송부터 일 문화. </li></ol> 흐름 사이토 분석을위한 5 준비 셀 <ol><li> 10 ml의 피펫과 15 ML 원뿔 튜브에 세포를 전송합니다. 5 ㎖의 PBS로 접시를 씻고 같은 원뿔 튜브로 전송할 수 있습니다. </li><li> 315 X g에서 펠렛 세포. 10 ml의 PBS와 펠렛을 씻으십시오. </li><li> 해동 세포 분리 용액 1 ㎖를 추가하고 가끔 교반과 함께 30 분 동안 37 ° C의 물을 욕조에 튜브를 배치합니다. 일부 세포 표면 항원의 검출을 위해, 트립신이 대신 사용될 수있다. </li><li> 1 ml의 차별화 미디어를 추가하고 최대 피펫 및 2 ml의 피펫과 함께 아래로. 도와 피펫의 끝 P200 팁을 사용하여 해체 단일 세포 현탁액으로 EB 소화. <ol><li> 세포막 단백질을 세포 표면에 재활용하기 위해서는 60 분 동안 다시 37 ° C의 물을 욕조에 튜브를 놓습니다. </li></ol></li><li> 5 분 동안 315 XG에서 세포 펠렛. </li><li> 세포 위스콘신 씻으일 0.1 % 소 혈청 알부민 분수 PBS (PBS / BSA)에서 제조 한 V (BSA)와 특정과 세포를 배양는 형광 항체를 태그. </li></ol> 조혈 전구 세포에 대한 6 유동 세포 계측법 분석 <ol><li> 5 ㎖의 12 × 75 라운드 MM 하단 튜브에 열 여섯 EB 파생 된 세포를 놓습니다. 보상 설정 사이토 흐름을 조절하기위한, 제조사의 지시에 따라 보상 비드를 사용한다. </li><li> 315 X g에서 펠렛 세포. 튜브 잔류 PBS / BSA 용액에 뜨는에 resuspend 펠렛을 붓고. </li><li> ESC의 HPC를 검출, 형광 표지 된 항체와 세포를 배양 : 항 - 마우스 CD41-PE (R-Phycoerytherin) 및 항 - 마우스 CD117 (cKit) -APC / CY7 (알로 피코시 아닌 7). <ol><li> 항체 1을 희석 : PBS / BSA 100을. 각 샘플에 희석 항체의 100 μl를 추가합니다. 각 공급 업체, 복제, 로트 또는 항체에 대한 항체의 정확한 희석을 결정합니다. 개별적으로 세포를 라벨에 대한 최적의 희석을 결정합니다. </리&gt; </ol></li><li> 30 분 동안 어둠 속에서 얼음에 샘플을 품어. </li><li> 5 분 동안 315 XG에 2 ㎖의 PBS / BSA 및 펠릿 세포를 추가합니다. </li><li> 기음 상층 액 300 ㎕의 PBS 또는 다른 등장 버퍼에 resuspend 펠렛. </li><li> 셀 스트레이너 캡 (0.35 μM)를 통해 필터 재현 탁 세포 대 세포 집합체를 제거합니다. </li><li> 플로우 사이토에 세포를 분석 할 수 있습니다. </li></ol>

<ol>
	<li>Williams, R. 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=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Desbaillets, I., Ziegler, U., Groscurth, P., Gassmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoid+bodies:+an+in+vitro+model+of+mouse+embryogenesis.">Embryoid bodies: an in vitro model of mouse embryogenesis.</a> Experimental physiology. 85, 645-651 (2000).</li><li>Hopfl, G., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+embryonic+stem+cells+into+embryoid+bodies.">Differentiating embryonic stem cells into embryoid bodies.</a> Methods Mol Biol. 254, 79-98 (2004).</li><li>Tian, X., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+towards+hematopoietic+cells:+progress+and+pitfalls.">Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.</a> Curr Opin Hematol. 15, 312-318 (2008).</li><li>Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+by+gene+targeting+in+mouse+embryo-derived+stem+cells.">Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.</a> Cell. 51, 503-512 (1987).</li><li>Mansour, S. L., Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+proto-oncogene+int-2+in+mouse+embryo-derived+stem+cells:+a+general+strategy+for+targeting+mutations+to+non-selectable+genes.">Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.</a> Nature. 336, 348-352 (1988).</li><li>Robertson, E., Bradley, A., Kuehn, M., Evans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+genes+introduced+into+cultured+pluripotential+cells+by+retroviral+vector.">Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector.</a> Nature. 323, 445-448 (1986).</li><li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells.">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li><li>Pfeifer, A., Ikawa, M., Dayn, Y., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+by+lentiviral+vectors:+lack+of+gene+silencing+in+mammalian+embryonic+stem+cells+and+preimplantation+embryos.">Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.</a> Proc Natl Acad Sci U S A. 99, 2140-2145 (2002).</li><li>Smith-Arica, J. 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=Infection+efficiency+of+human+and+mouse+embryonic+stem+cells+using+adenoviral+and+adeno-associated+viral+vectors.">Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors.</a> Cloning and stem cells. 5, 51-62 (2003).</li><li>Lakshmipathy, U., 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+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Cook, B. D., Liu, S., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad1+signaling+restricts+hematopoietic+potential+after+promoting+hemangioblast+commitment.">Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment.</a> Blood. 117, 6489-6497 (2011).</li><li>Zafonte, B. T., 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=Smad1+expands+the+hemangioblast+population+within+a+limited+developmental+window.">Smad1 expands the hemangioblast population within a limited developmental window.</a> Blood. 109, 516-523 (2007).</li><li>Kyba, M., Perlingeiro, R. C., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HoxB4+confers+definitive+lymphoid-myeloid+engraftment+potential+on+embryonic+stem+cell+and+yolk+sac+hematopoietic+progenitors.">HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.</a> Cell. 109, 29-37 (2002).</li><li>Galvin, K. E., Travis, E. D., Yee, D., Magnuson, T., Vivian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+signaling+regulates+the+bone+morphogenic+protein+pluripotency+pathway+in+mouse+embryonic+stem+cells.">Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.</a> J Biol Chem. 285, 19747-19756 (2010).</li><li>Ismailoglu, I., Yeamans, G., Daley, G. Q., Perlingeiro, R. C., Kyba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+patterning+activity+of+SCL.">Mesodermal patterning activity of SCL.</a> Exp Hematol. 36, 1593-1603 (2008).</li><li>Kyba, M., 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=Enhanced+hematopoietic+differentiation+of+embryonic+stem+cells+conditionally+expressing+Stat5.">Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.</a> Proc Natl Acad Sci U S A. 100, 11904-11910 (2003).</li><li>Cao, M., 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=MiR-23a+regulates+TGF-beta-induced+epithelial-mesenchymal+transition+by+targeting+E-cadherin+in+lung+cancer+cells.">MiR-23a regulates TGF-beta-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells.</a> Int J Oncol. 41, 869-875 (2012).</li><li>Chan, M. 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=Molecular+basis+for+antagonism+between+PDGF+and+the+TGFbeta+family+of+signalling+pathways+by+control+of+miR-24+expression.">Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression.</a> EMBO J. 29, 559-573 (2010).</li><li>Huang, 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=Upregulation+of+miR-23a+approximately+27a+approximately+24+decreases+transforming+growth+factor-beta-induced+tumor-suppressive+activities+in+human+hepatocellular+carcinoma+cells.">Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells.</a> Int J Cancer. 123, (2008).</li><li>Min, 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=TGF-beta-associated+miR-27a+inhibits+dendritic+cell-mediated+differentiation+of+Th1+and+Th17+cells+by+TAB3,+p38+MAPK,+MAP2K4+and+MAP2K7.">TGF-beta-associated miR-27a inhibits dendritic cell-mediated differentiation of Th1 and Th17 cells by TAB3, p38 MAPK, MAP2K4 and MAP2K7.</a> Genes Immun. 13, 621-631 (2012).</li><li>Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD41+marks+the+initiation+of+definitive+hematopoiesis+in+the+mouse+embryo.">Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.</a> Blood. 101, 508-516 (2003).</li><li>Mitjavila-Garcia, M. T., 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=Expression+of+CD41+on+hematopoietic+progenitors+derived+from+embryonic+hematopoietic+cells.">Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.</a> Development. 129, 2003-2013 (2002).</li><li>Warr, M. R., Pietras, E. M., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+hematopoietic+stem+cell+functions+during+normal+hematopoiesis+and+hematological+malignancies.+Wiley+interdisciplinary+reviews.">Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies. Wiley interdisciplinary reviews.</a> Systems biology and medicine. 3, 681-701 (2011).</li><li>Dahl, R., Ramirez-Bergeron, D. L., Rao, S., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spi-B+can+functionally+replace+PU.1+in+myeloid+but+not+lymphoid+development.">Spi-B can functionally replace PU.1 in myeloid but not lymphoid development.</a> Embo J. 21, 2220-2230 (2002).</li><li>Dahl, 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=Regulation+of+macrophage+and+neutrophil+cell+fates+by+the+PU.1:C/EBPalpha+ratio+and+granulocyte+colony-stimulating+factor.">Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor.</a> Nat Immunol. 4, 1029-1036 (2003).</li></ol>

저자는 공개 경쟁 금융 이익을 더이 없습니다.

상술 한 바와 같이, inducibly 관심 유전자를 발현 ESC 클론 독시사이클린 시스템을 사용하여 생성 될 수 있지만,이 라인의 생성 시간 소모적이고 노동 집약적이다. 이 프로토콜에서 설명하는 교환 사채 파생 ESC로부터 제조 된 단일 세포 현탁액에 대한 관심의 유전자를 표현하는 방법이다. 이 감염된 세포는 이후의 차별화를 검사 할 수 드롭을 걸어서 사채로 개편된다. 예를 (그림 3)에서는</strong...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

<table><tbody><tr><td>DMEM</td><td>Sigma/Aldrich</td><td>D5796</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma/Aldrich</td><td>M3148</td><td></td></tr><tr><td>FBS (ES Screened)</td><td>Hyclone</td><td>SH30070.03E</td><td></td></tr><tr><td>FBS (Defined)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>Penn/Strep</td><td>Life Technologies</td><td>15070</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>LIF ESGRO</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>ACCUMAX</td><td>Millipore</td><td>SCR006</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Falcon Non-treated Plates 6-well</td><td>Fisher Scientific</td><td>08-772-49</td><td></td></tr><tr><td>Falcon Petri dish 10 cm</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Falcon Petri dish 15 cm</td><td>Fisher Scientific</td><td>08-757-148</td><td></td></tr><tr><td>Falcon Tube 5 ml</td><td>Fisher Scientific</td><td>14-959-11A</td><td></td></tr><tr><td>Falcon Tube 5 ml with Cell Strainer Cap</td><td>Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>White sterile resevoirs</td><td>U.S.A. Scientific</td><td>111-0700</td><td></td></tr><tr><td>Rat anti-mouse CD41 PE-conjugated</td><td>Biolegend</td><td>133906</td><td></td></tr><tr><td>Rat anti-mouse CD117 APC/CY7-conjugated</td><td>Biolegend</td><td>105826</td><td></td></tr><tr><td>RW4 ESCs</td><td>ATCC</td><td>CRL-12418</td><td></td></tr></tbody></table>

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

RW4을 젤라틴이 연구에서 사용 된 ESCS (129X1 / SVJ 마우스 변형에서 파생). 교환 사채는 차별화 삼일에 고립되었다. 최상의 결과를 위해 사채는 구형 및 비 부착 (그림 1A, B)해야한다. 바이러스가 관심의 유전자와 함께 형광 마커 GFP를 공동으로 표현하는 spinoculation 후, 교환 사채는 매달려 드롭 방식으로 개혁되었다. 사채 성공적 2.0, 2.5, 및 3.0 일 사채로부터 제조 세포 현탁액으로부터 개질...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

쥐과 배아의 레트로 바이러스 감염은 조혈 분화 분석을위한 세포 유래 배아 바디 줄기 세포

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Research

JoVE Journal

Biology

8.4K 조회수.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 

비디오: Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

연구

생물학

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

쥐 배아 Hemogenic 내피 세포의 분리

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

발생학

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

두와 입체 배아 체에서 마우스 배아 줄기 세포의 레티노 산에 의한 신경 분화 분석

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

골수 전구 세포의 레트로 바이러스 형질 도입은 T 세포 수용체 Retrogenic 마우스를 생성하는

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

면역학 및 감염병학

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

인간화 된 마우스 모델에 HIV 감염에 대 한 줄기 세포를 기반으로 설계된 내성

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

의학

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

질병 모델에서 조혈 세포의 연구를 위한 렌티바이러스 CRISPR/Cas9 매개 게놈 편집

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

줄기 세포 유래 바이러스 Ag 특이적 T 림프구는 마우스에서 HBV 복제를 억제

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

뮤린 B-1a 세포에 CXCR4의 레트로 바이러스 과발현 및 골수와 IgM 생산에 표적 B-1a 세포 이동을 위한 채택 전송

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...