Name: busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice
Uploaded: 2015-04-01T14:00:00.000+00:00
Duration: 11 min 25 s
Description: Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

This work was supported by the Ronald Peter Griggs Memorial Fellowship in ALS Research (to KP) and a Neuromuscular Research Partnership Program grant from the CIHR, the ALS Society of Canada, and Muscular Dystrophy Canada (JNM-69682) to CK and FMR. Additional support was also provided by the ALS Society of America (57969). We would like to thank Dr. R. Keith Humphries from the Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, Canada and the staff at the Simon Fraser University Animal Care Unit.

참고 : 윤리 성명 :이 프로토콜을 검토하고 사이먼 프레이저 대학의 동물 관리위원회의 승인을받은 (UACC, 허가 번호 1037K-12와 1060K-03) 및 동물 관리, NIH 가이드에 캐나다 협의회 준수 실험실 동물의 관리 및 사용 및 EEC 이사회 지침. 1. 특별 고려 사항 참고 : 술판은 세포 독성이다. 핸들과 물질 안전 보건 자료 및 제도적 지침에 따라 폐기하십시오. <ol><li> 층류 후드에서 무균 기술을 모두 수행한다. </li><li> 이러한 박리 패키지, 오토 클레이브와 같은 적절한 포장 재료에 포장하여 사용 전에기구를 멸균. </li><li> 감염 위험이 조사에 비해 술판 컨디셔닝 낮지 만 이식 후 처음 2 주 층류 후드에서 동물을 처리한다. </li></ol> 2. 컨디셔닝받는 사람 마우스 <ol><li> 멸균 인산 완충 식염수 (PBS)와 ml의 3 ㎎ /에 술판을 희석. 참고 : 주입 매일 사용하기 직전에 부설 판의 신선한 희석을하는 것이 중요합니다. </li><li> 매일 IP 주입을 통해받는 사람 마우스에 희석 부설 판의 20 ㎎ / ㎏을 관리합니다. </li><li> 60 ~ 100 mg의 총 투여 량까지 20 ㎎ / ㎏ 부설 판의 일상 IP 주사를 반복 / kg (즉, 80 ㎎ / ㎏ 총 복용량, 4 일 연속 부설 판의 20 ㎎ / ㎏을 투여) 전달되었습니다. </li></ol> 기증자 골수 세포의 3. 분리 참고 :이 프로토콜은 성공적으로 5 기증자 마우스로부터 BM 세포를 분리하고 제조에 사용되었습니다. 일반적으로 마우스 당 세포 수율은 12 ~ 16받는 사람 쥐를 이식하기에 충분 약 천 -4 천만 BMDCs입니다. 이상의 공여 쥐 필요하다면 프로토콜 따라서 조정될 필요가있다. <ol><li> 술판 조절 euth의 마지막 날 다음CO 2를 사용하여 (1-6개월 이전) GFP 기증자 마우스를 anize (또는 기관에서 받아 다른 안락사 절차에 의해). 이식 합병증받는 사람과 같은 성이다 동계 기증자를 사용하지 않도록합니다. </li><li> 70 % 에탄올로 마우스 스프레이. 복부 및 사용하여 수술 가위에서 리프트 피부까지 복강 발목을 향해 다리에서 피부를 통해 절개를합니다. 발을 잡고 단단히 다리 조직을 노출 엉덩이쪽으로 발목으로부터 피부를 잡아 당깁니다. </li><li> 고관절을 노출 대퇴골에서 근육과 지방 조직을 멀리 트림. </li><li> 부드럽게 다리를 확장 도보로 당기면서, 고관절에 가위를 누릅니다. 그냥 대퇴골 자체를 절단하지 대퇴골 복용 치료의 머리 위에 잘라. </li><li> , 불임을 유지 도보로 다리를 잡고 멸균 조직과 뼈의 표면을 문질러 뼈에 남아있는 모든 조직을 청소 도움이됩니다. </li><li> 무릎 관절을 통해 절단에 의해 대퇴골과 경골을 분리하고멸균 PBS를 포함하는 배양 접시에서 대퇴골을 배치합니다. 얼음에 품어. </li><li> 제거하고 비골은 경골에 연결되는 지점에서 절단하여 비골을 폐기합니다. 대퇴골과 배양 접시에서 경골를 놓고 얼음에 품어. </li><li> 반복하여 다른 다리에 대한 3.2-3.7 단계, 그리고 필요한 경우, 추가 기증자 마우스. 뼈를 제거한 다음, 뜨거운 비드 살균기와 수술 도구를 소독 또는 후속 단계에 대한 멸균 새로운 도구를 사용합니다. </li><li> 대퇴골의 경우, 집게와 대퇴골을 잡고 조심스럽게 &#39;면도&#39;수술 가위를 사용하여 말단 뼈를 종료합니다. BM 구멍을 노출하는 필요에 따라 뼈의 적은을 제거합니다. </li><li> 멸균 PBS의 3 ㎖ 주사기를 입력하고 23 G 바늘을 연결합니다. 조심스럽게 BM 구멍에 바늘을 지루하게하고 멸균 배양 접시에 BM을 세척하십시오. 원하는 모든 세포의 제거를 보장하기 위해 바늘 포인트와 수질 공동을 긁어해야합니다. 추출 후,빨간 BM은 더 이상 볼 수없고 뼈가 지금 흰색 나타나는지 확인합니다. </li><li> 반복 같은 배양 접시에 BM의 모든 풀링, 이후 대퇴골에 대한 3.9-3.10 단계를 반복합니다. </li><li> 경골를 들어, 조심스럽게 집게로 경골과 &#39;면도&#39;경골이 BM 캐비티를 노출 무릎에 부착 된 끝을 잡고. 적색 가시 BM이 끝나는 뼈를 따라 두 번째 컷을 확인합니다. </li><li> 멸균 PBS의 3 ㎖ 주사기를 입력하고 25 G 바늘을 연결합니다. 조심스럽게 (무릎에 부착 된 끝에서) BM 구멍에 바늘을 삽입하고 대퇴골에서 BM을 포함하는 배양 접시에 BM을 세척하십시오. 모든 세포를 제거하기 위해 바늘 BM 공동의 벽을 긁어. 추출 후, 빨간색 BM은 더 이상 볼 수없고 뼈가 지금 흰색 나타나는지 확인합니다. </li><li> 반복 같은 배양 접시에 BM의 모든 풀링, 이후의 경골에 대한 3.12-3.13 단계를 반복합니다. </li><li> 부드럽게 C를 해리 1 ML의 피펫 팁과 BM을 씹다ELL 학생. </li><li> 멸균 50 ㎖ 원심 분리 튜브에 40 μM 바스켓 필터를 통해 세포 현탁액 합격. 남아있는 세포를 얻을 원심 분리 튜브에 필터를 통해이를 전송하는 PBS로 접시를 씻어. </li><li> 4 ° C에서 450 XG에 5 분 동안 원심 분리기. 제거하고 상층 액을 버린다. </li><li> 적혈구 용해하는 버퍼의 3 ㎖의 펠렛을 다시 일시 중지합니다. 8.5 분 동안 얼음에서 인큐베이션. </li><li> 용균 버퍼를 해소하기 위해 멸균 PBS의 ~ 30 ML을 추가합니다. </li><li> 4 ° C에서 450 XG에 5 분 동안 원심 분리기. 제거하고 상층 액을 버린다. </li><li> 멸균 PBS 1 ㎖에 펠릿을 다시 일시. 얼음에 보관하십시오. </li><li> 세포 균질 작은 분취 량을 가지고 PBS로 희석 (1 : 100 1 마우스 1 : 200이 쥐 등)를 이용하여 혈구 세포 수를 정량화. </li><li> 8 × 106 세포 / ml에 세포 현탁액을 희석시키고, 얼음에 주입 될 때까지 배양한다. </li></ol> 4. 골수 이식 <ol><li> 인터넷LL 각 마우스에 대한 희석 세포 현탁액 300 μL와 (28 G 인슐린 바늘) 0.5 ML의 주사기를 주입한다. 주사기에 존재하는 공기 방울을 제거해야합니다. </li><li> 가열 패드 컨디셔닝받는 마우스를 함유하는 마우스 케이지를두고 꼬리 정맥이 팽창 할 수있다. </li><li> 구속 장치에서받는 마우스와 장소를 가져 가라. 70 % 에탄올로 적셔 수술 거즈와 꼬리를 닦습니다. </li><li> 단단히 꼬리를 잡고 경사 위로 부드럽게 측면 꼬리 정맥 중 하나에 바늘을 삽입하고 세포 현탁액을 주입. </li><li> 출혈이 새로운 깨끗한 케이지에 마우스를 도입하기 전에 멈출 때까지 주사 부위에 수술 거즈를 잡습니다. </li></ol> 5. BM 키메라의 정도를 추정 주 : 키메라 수준 일주 BMT 이후에 말초 혈액에서 통상적 변수이며, 이러한 키메라 레벨은​​ 통상 적어도 이주 BMT 이후로 추정된다. Chim은의 수준erism 포스트 BMT 3-4주 내 말초 혈액에서&gt; 80 % GFP + 세포에 도달해야한다. <ol><li> FACS 완충액 (PBS 2 mM의 EDTA + 2 % 소 태아 혈청)을 준비한다. 주 : FACS 완충액 몇 주 동안 39 ° C에서 저장 될 수있다. </li><li> 금지 튜브에 마우스를 놓고 측면 복재 정맥을 노출 후방 다리에 털을 면도. </li><li> 얇게 코트 석유 젤리와 피어스 25 G 바늘과 복재 정맥과 다리. </li><li> 헤파린 코팅 된 모세관에 혈액의 약 50 μl를 수집합니다. FACS 완충액 1 ㎖를 함유하는 마이크로 원심 분리 튜브로 혈액을 추가한다. 반전에 의해 튜브를 혼합하고 얼음에 저장합니다. </li><li> 다시 케이지에 마우스를 반환하기 전에 정지 출혈까지 주사 부위에 수술 거즈를 잡습니다. </li><li> X 900 g에서 5 분 동안 혈액 샘플을 원심 분리기. 제거하고 상층 액을 버린다. </li><li> 적혈구 용혈 버퍼 500 μL에 펠렛을 다시 일시 중지합니다. 8.5 분 동안 얼음에서 인큐베이션. </li><li> 용균 버퍼를 해소하기 위해 FACS 버퍼의 1 ML을 추가합니다. </li><li> g X 900에서 5 분 동안 원심 분리. 제거하고 상층 액을 버린다. </li><li> 적혈구 용혈 버퍼 500 μL에 두 번째 용해 단계를 수행합니다. 8.5 분 동안 얼음에서 인큐베이션. </li><li> 용균 버퍼를 해소하기 위해 FACS 버퍼의 1 ML을 추가합니다. </li><li> g X 900에서 5 분 동안 원심 분리. 제거하고 상층 액을 버린다. 펠렛은 이제 흰색 있는지 확인합니다. 주 : 펠렛이 여전히 적색 나타나면 추가적인 분해 단계는 다음 단계로 진행하기 전에 필요할 수있다. </li><li> FACS 완충액 1 ㎖에서 펠릿을 다시 일시. g X 900에서 5 분 동안 원심 분리. 제거하고 상층 액을 버린다. </li><li> FACS 완충액 300 μL의 펠릿을 다시 중지하고 유세포 사용 GFP + 세포를 정량화 (주 :이 시점에서 셀로부터 임의의 면역 염색은 표준 기술을 이용하여 수행 될 수 있으며, 종래는 유세포 분석 유동). </li></ol>

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	<li>Ginhoux, F., 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=Fate+Mapping+Analysis+Reveals+That+Adult+Microglia+Derive+from+Primitive+Macrophages.">Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Hickey, W. F., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+microglial+cells+of+the+CNS+are+bone+marrow-derived+and+present+antigen+in+vivo.">Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.</a> Science (New York, N. Y.). 239 (4837), 290-292 (1988).</li><li>Duran-Struuck, R., Dysko, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+bone+marrow+transplantation+(BMT):+providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 48 (1), 11-22 (2009).</li><li>Jimenez, M., Ercilla, G., Martinez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+reconstitution+after+allogeneic+stem+cell+transplantation+with+reduced-intensity+conditioning+regimens.">Immune reconstitution after allogeneic stem cell transplantation with reduced-intensity conditioning regimens.</a> Leukemia. 21 (8), 1628-1637 (2007).</li><li>Ellen Nath, C., John Shaw, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Busulphan+in+blood+and+marrow+transplantation:+dose,+route,+frequency+and+role+of+therapeutic+drug+monitoring.">Busulphan in blood and marrow transplantation: dose, route, frequency and role of therapeutic drug monitoring.</a> Current clinical pharmacology. 2 (1), 75-91 (2007).</li><li>Galaup, A., Paci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+dimethanesulfonate+alkylating+agents:+busulfan+and+treosulfan.">Pharmacology of dimethanesulfonate alkylating agents: busulfan and treosulfan.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 9 (3), 333-347 (2013).</li><li>Lewis, C. -. A. 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=Myelosuppressive+Conditioning+Using+Busulfan+Enables+Bone+Marrow+Cell+Accumulation+in+the+Spinal+Cord+of+a+Mouse+Model+of+Amyotrophic+Lateral+Sclerosis.">Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis.</a> PLoS ONE. 8 (4), e60661 (2013).</li><li>Capotondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+conditioning+is+instrumental+for+successful+microglia+reconstitution+following+hematopoietic+stem+cell+transplantation.">Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation.</a> Proceedings of the National Academy of Sciences. 109 (37), 15018-15023 (2012).</li><li>Wilkinson, F. L., Sergijenko, A., Langford-Smith, K. J., Malinowska, M., Wynn, R. F., Bigger, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Busulfan+Conditioning+Enhances+Engraftment+of+Hematopoietic+Donor-derived+Cells+in+the+Brain+Compared+With+Irradiation.">Busulfan Conditioning Enhances Engraftment of Hematopoietic Donor-derived Cells in the Brain Compared With Irradiation.</a> Molecular Therapy. 21 (4), 868-876 (2013).</li><li>Lampron, A., Pimentel-Coelho, P. M., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+bone+marrow-derived+cells+into+the+CNS+in+models+of+neurodegeneration:+Naturally+Occurring+Migration+of+BMDC+into+the+CNS.">Migration of bone marrow-derived cells into the CNS in models of neurodegeneration: Naturally Occurring Migration of BMDC into the CNS.</a> Journal of Comparative Neurology. , 3863-3876 (2013).</li><li>Kierdorf, K., Katzmarski, N., Haas, C. A., Prinz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow+Cell+Recruitment+to+the+Brain+in+the+Absence+of+Irradiation+or+Parabiosis+Bias.">Bone Marrow Cell Recruitment to the Brain in the Absence of Irradiation or Parabiosis Bias.</a> PLoS ONE. 8 (3), e58544 (2013).</li><li>Ajami, B., Bennett, J. L., Krieger, C., McNagny, K. M., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltrating+monocytes+trigger+EAE+progression,+but+do+not+contribute+to+the+resident+microglia+pool.">Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.</a> Nature Neuroscience. 14 (9), 1142-1149 (2011).</li><li>Down, J. D., Tarbell, N. J., Thames, H. D., Mauch, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+and+allogeneic+bone+marrow+engraftment+after+total+body+irradiation:+dependence+on+dose,+dose+rate,+and+fractionation.">Syngeneic and allogeneic bone marrow engraftment after total body irradiation: dependence on dose, dose rate, and fractionation.</a> Blood. 77 (3), 661-669 (1991).</li><li>Van Pel, M., van Breugel, D. W. J. G., Vos, W., Ploemacher, R. E., Boog, C. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+myeloablative+regimen+with+clinical+potential:+I.+Treosulfan+conditioning+and+bone+marrow+transplantation+allow+induction+of+donor-specific+tolerance+for+skin+grafts+across+full+MHC+barriers.">Towards a myeloablative regimen with clinical potential: I. Treosulfan conditioning and bone marrow transplantation allow induction of donor-specific tolerance for skin grafts across full MHC barriers.</a> Bone Marrow Transplantation. 32 (1), 15-22 (2003).</li><li>Ajami, B., Bennett, J. L., Krieger, C., Tetzlaff, W., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nature Neuroscience. 10 (12), 1538-1543 (2007).</li><li>Andersson, B. 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=Conditioning+therapy+with+intravenous+busulfan+and+cyclophosphamide+(IV+BuCy2)+for+hematologic+malignancies+prior+to+allogeneic+stem+cell+transplantation:+a+phase+II+study.">Conditioning therapy with intravenous busulfan and cyclophosphamide (IV BuCy2) for hematologic malignancies prior to allogeneic stem cell transplantation: a phase II study.</a> Biology of Blood and Marrow Transplantation. 8 (3), 145-154 (2002).</li><li>Hassan, 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=Cerebrospinal+fluid+and+plasma+concentrations+of+busulfan+during+high-dose+therapy.">Cerebrospinal fluid and plasma concentrations of busulfan during high-dose therapy.</a> Bone Marrow Transplantation. 4 (1), 113-114 (1989).</li><li>Hassan, 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=In+vivo+distribution+of+[11C]-busulfan+in+cynomolgus+monkey+and+in+the+brain+of+a+human+patient.">In vivo distribution of [11C]-busulfan in cynomolgus monkey and in the brain of a human patient.</a> Cancer Chemotherapy and Pharmacology. 30 (2), 81-85 (1992).</li><li>Jung, 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=Analysis+of+Fractalkine+Receptor+CX3CR1+Function+by+Targeted+Deletion+and+Green+Fluorescent+Protein+Reporter+Gene+Insertion.">Analysis of Fractalkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluorescent Protein Reporter Gene Insertion.</a> Molecular and Cellular Biology. 20 (11), 4106-4114 (2000).</li><li>Harrison, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+repopulation:+a+new+assay+for+long-term+stem+cell+functional+capacity.">Competitive repopulation: a new assay for long-term stem cell functional capacity.</a> Blood. 55 (1), 77-81 (1980).</li><li>Evans, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+intravital+imaging+reveals+that+blood-derived+macrophages+but+not+resident+microglia+facilitate+secondary+axonal+dieback+in+traumatic+spinal+cord+injury.">High-resolution intravital imaging reveals that blood-derived macrophages but not resident microglia facilitate secondary axonal dieback in traumatic spinal cord injury.</a> Experimental Neurology. 254, 109-120 (2014).</li></ol>

The authors have nothing to disclose.

술판 조절은 쉽게 감지 할 수 있습니다 조혈 세포와 생쥐에서 높은 수준의 BM의 키메라의 생성을 허용합니다. 이러한 조절은, 조사 설비를 필요로하지 않고 행할 수있다. 더욱이,이 프로토콜에 설명 술판의 투여 량으로 골수 억제는 잘 조사 골수 투여에 의한 독성 부작용을 최소화 허용하고, 따라서 더 많은 아르 젊은 오래 또는 병에 걸린 마우스에서 BM의 키메라를 생성하기 위해 더 적합한 기술 ?...

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis1. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals1, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the &#946;-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays. 
For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called &#8216;lethal irradiation&#8217;). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections3. As such, reduced intensity conditioning (so called &#8216;RIC&#8217; protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly4. 
Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab7, as well as in several other recent publications8&#8211;11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM7. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation7. 
We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)7 and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders7&#8211;11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning7, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.
While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

<table><tbody><tr><td>Busulfex</td><td>Otsuka Canada Pharmaceutical Inc.</td><td>DIN 02240602</td><td>Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.</td></tr><tr><td>ACK Lysing Buffer</td><td>Life Technologies</td><td>A10492-01</td><td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>12483-020</td><td></td></tr></tbody></table>

busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

60 ~ 100 mg을 투여 / 부설 판의 kg은 일반적으로 잘 마우스에 의해 허용됩니다. 그러나 동물은 일반적으로 식습관을 격려하기 위해 조사 해바라기 씨 등의 치료와 보완 될 필요가있다 조절 단계 (~ 5 ~ 10 %) 때문에 다이어트 중에 체중을 잃게됩니다. 말초 혈액에서&gt; 80 % GFP + 세포의 키메라와 BM의 수준은 60 ~ 100 ㎎ / ㎏ 부설 판 (7)을 사용하여 지속적으로 달성해야한다. 우리의 실험은 성공적으...

Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

마우스에서의 높은 수준의 안정적인 골수 키메라를 생성하기위한 에이전트로서 골수 억제 술판

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

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13.2K 조회수.  Simon Fraser University. We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

비디오: Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

골수 증식 종양의 전달-이식 마우스 모델

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

연구

암 연구학

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

인간 말초 혈액 단핵 세포 (PBMC) 번역 면역 종양학에 대한 인간화 제종이식 모델 (I-O) 연구

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

세포의 기원, 종류, 그리고 상호 작용의 시각화를위한 형광 조직 이식에 따라 정량 멀티 스펙트럼 분석

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

의학

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

조 혈 줄기 세포 이식 Myelodysplastic 증후군의 평가에 사용

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

발생학

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

급성 림프구성 백혈병 환자 유래 이종이식식 마우스 모델을 이용한 키메라 항원 수용체 T 세포 관련 독성 평가

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

라이브 쥐의 대퇴부 골수 흡인

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

면역학 및 감염병학

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

이식대 숙주 질환에서 수지상 세포의 역할을 조사하는 골수 이식 플랫폼

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

<meta charset="utf8"></head><body>살아있는 쥐의 골수 연구를 위한 대퇴 내 주사

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>살아있는 마우스를 사용한 간소화된 대퇴강 주사로 지속적인 골수 분석 가능

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

살아있는 마우스를 사용한 간소화된 대퇴강 주사로 지속적인 골수 분석 가능

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...