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.

NOTA: Ética Declaração: Este protocolo foi revisto e aprovado pelo Comitê de Cuidados Animal da Simon Fraser University (UACC; permitir números 1037K-12 e 1060K-03) e está em conformidade com a Canadian Council on Animal Care, o Guia do NIH para Cuidados e Uso de Animais de Laboratório, e da Directiva do Conselho CEE. 1. Considerações Especiais NOTA: O bussulfano é citotóxica. Manusear e eliminar de acordo com a ficha de segurança e diretrizes institucionais. <ol><li> Executar todas as técnicas de forma asséptica numa campânula de fluxo laminar. </li><li> Esterilizar instrumentos antes da sua utilização por envolvimento em um material de embalagem adequado, tal como um pacote de casca, e autoclavagem. </li><li> Embora o risco de infecção é baixo comparado com o condicionamento de busulfano a irradiação, apenas tratar os animais numa câmara de fluxo laminar para as primeiras 2 semanas após o transplante. </li></ol> 2. Condicionamento deOs ratinhos receptores <ol><li> Diluir busulfan, a 3 mg / ml com solução salina tamponada com fosfato estéril (PBS). NOTA: É importante fazer uma nova diluição de busulfan imediatamente antes de usar cada dia da injeção. </li><li> Administrar 20 mg / kg de busulfano diluída para murganhos receptores por via de injecção IP diariamente. </li><li> Repita injecções IP diárias de 20 mg / kg de busulfano até uma dose total de 60-100 mg / kg foi administrada (isto é, para um 80 mg / kg dose total, administrar 20 mg / kg de busulfano durante 4 dias consecutivos). </li></ol> 3. Isolamento de Doadores de Medula Óssea Cells NOTA: Este protocolo tem sido utilizado com sucesso para isolar e preparar a partir de células de MO até 5 ratinhos dadores. Normalmente, o rendimento das células por rato é de aproximadamente 30-40000000 BMDCs, o que é suficiente para transplantar 16/12 ratos destinatário. Se forem necessários mais ratos doadores o protocolo pode precisar de ser ajustado em conformidade. <ol><li> Após o último dia de busulfan condicionado euthanize um rato doador GFP (1-6 meses de idade), utilizando CO 2 (ou de qualquer outro procedimento de eutanásia aceitos na instituição). Para evitar complicações de enxerto utilizar doadores singeneicas que são do mesmo sexo, como os destinatários. </li><li> Spray de rato com etanol 70%. Elevador pele nas tesouras cirúrgicas abdómen e utilizando-se a incisão através da pele a partir da cavidade abdominal para cima da perna para o tornozelo. Segurando o pé, firmeza da pele a partir do tornozelo para o quadril expondo o tecido perna. </li><li> Apare músculo e tecido adiposo do fêmur para expor a articulação do quadril. </li><li> Enquanto puxa suavemente no pé para estender a perna, pressione a tesoura contra a articulação do quadril. Corte logo acima da cabeça do fêmur tomando cuidado para não cortar o próprio fêmur. </li><li> Para ajudar a manter a esterilidade, segure a perna pelo pé e limpe qualquer tecido restante dos ossos, esfregando a superfície do osso com tecidos autoclavada. </li><li> Separe o fêmur e da tíbia, cortando através da articulação do joelho ecoloque o fêmur em uma placa de cultura contendo PBS estéril. Incubar em gelo. </li><li> Retire e elimine a fíbula por corte nos pontos em que a fíbula se conecta à tíbia. Coloque a tíbia do prato de cultura com o fémur, e incubar em gelo. </li><li> Repita os passos de 3,2-3,7 para a outra perna, e se necessário, dos ratinhos dadores adicionais. Após a remoção dos ossos, esterilizar os instrumentos cirúrgicos com um esterilizador quente talão ou usar um novo conjunto de ferramentas estéreis para as etapas subseqüentes. </li><li> Para os fêmures, segure o fêmur com uma pinça e usando uma tesoura cirúrgica cuidadosamente &quot;shave&quot; as extremidades distais da osso. Remover o mínimo do osso como necessário para expor a cavidade BM. </li><li> Encha uma seringa com 3 ml de PBS estéril e anexar uma agulha G 23. Suportou cuidadosamente a agulha na cavidade BM e lave o BM em um prato de cultura estéril. Certifique-se de raspar a cavidade medular com a ponta da agulha para assegurar a remoção de todas as células desejadas. Após a extracção,assegurar que o BM vermelho já não é visível e o osso agora parece branca. </li><li> Repita os passos 3,9-3,10 para fêmures posteriores, reunindo todo o BM no mesmo prato de cultura. </li><li> Para as tíbias, segure a tíbia com uma pinça e cuidadosamente &quot;shave&quot; o final, onde a tíbia foi anexado ao joelho para expor a cavidade BM. Faça um segundo corte ao longo do osso onde o BM vermelho visível termina. </li><li> Encha uma seringa com 3 ml de PBS estéril e anexar uma agulha G 25. Inserir a agulha suavemente para dentro da cavidade BM (a partir da extremidade que foi ligado ao joelho) e lave o BM para o prato de cultura contendo o BM a partir dos fémures. Raspe as paredes da cavidade BM com a agulha para remover todas as células. Após a extracção, garantir que o BM vermelho não é mais visível e o osso agora aparece branco. </li><li> Repita os passos de 3,12-3,13 para tíbias subsequente, reunindo todo o BM no mesmo prato de cultura. </li><li> Delicadamente, triturar o BM com uma ponteira de 1 ml para dissociar a cvaras. </li><li> Passar a suspensão de células através de um filtro de carrinho 40 uM em um tubo de centrífuga de 50 mL estéril. Lave o prato com PBS para obter todas as células restantes e transferir este através do filtro no tubo de centrífuga. </li><li> Centrifugar durante 5 minutos a 450 xg, a 4 ° C. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 3 ml de tampão de lise de eritrócitos. Incubar em gelo durante 8,5 min. </li><li> Adicionar ~ 30 ml de PBS estéril para saciar o tampão de lise. </li><li> Centrifugar durante 5 minutos a 450 xg, a 4 ° C. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 1 ml de PBS estéril. Mantenha no gelo. </li><li> Tomar uma pequena aliquota do homogeneizado de células e dilui-se com PBS (1: 100 para 1 de ratinho; 1: 200 durante 2 murganhos, etc.) para quantificar o número de células utilizando um hemocitómetro. </li><li> Dilui-se a suspensão de células a 8 x 10 6 células / ml e incuba-se em gelo até injecção. </li></ol> 4. Transplante de Medula Óssea <ol><li> Fill uma seringa de 0,5 ml (com uma agulha de insulina 28 L) com 300 uL de suspensão diluída de células para cada ratinho a ser injectado. Certifique-se de remover todas as bolhas de ar presentes na seringa. </li><li> Coloque a gaiola do rato contendo os ratinhos receptores condicionados numa almofada de aquecimento e permita que as veias da cauda para dilatar. </li><li> Tome um rato destinatário e lugar no dispositivo de retenção. Limpe a cauda com gaze cirúrgica embebido com álcool 70%. </li><li> Segurar firmemente a cauda e com o bisel para cima, inserir a agulha suavemente numa das veias da cauda laterais e injectar a suspensão de células. </li><li> Segurar gaze cirúrgica no local da injecção até o sangramento pára antes de introduzir o rato em uma gaiola limpa novo. </li></ol> 5. estimativa da extensão da BM quimerismo NOTA: Os níveis de quimerismo são tipicamente variável no sangue periférico em uma semana pós-TMO, e, como tal, os níveis de quimerismo são normalmente estimada pelo menos 2 semanas de pós-TMO. Níveis de chimerism deve atingir&gt; 80% GFP + células no sangue periférico dentro de 3-4 semanas pós-TMO. <ol><li> Preparar tampão de FACS (2 mM de EDTA + 2% de soro fetal de bovino em PBS). NOTA: tampão FACS pode ser armazenado a 4 ° C durante várias semanas. </li><li> Posicione o mouse em um tubo de restrição e raspar a pele na perna posterior para expor a veia safena lateral. </li><li> Coat fina da perna com vaselina e perfuram a veia safena com uma agulha 25 G. </li><li> Recolhe aproximadamente 50 ul de sangue num tubo capilar com heparina revestidos. Adicionar o sangue para um tubo de micro-centrifugação contendo 1 ml de tampão de FACS. Misturar por inversão do tubo e armazenar em gelo. </li><li> Segure gaze cirúrgica no local da injeção, até o sangramento parar antes de retornar mouse de volta para a jaula. </li><li> Centrifuga-se as amostras de sangue durante 5 minutos a 900 x g. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 500 mL de tampão de lise de eritrócitos. Incubar em gelo durante 8,5 min. </li><li> Adicionar 1 mL de tampão de FACS para extinguir o tampão de lise. </li><li> Centrifuga-se as amostras durante 5 min a 900 x g. Retire e elimine o sobrenadante. </li><li> Executar um segundo passo de lise em 500 uL de tampão de lise de eritrócitos. Incubar em gelo durante 8,5 min. </li><li> Adicionar 1 mL de tampão de FACS para extinguir o tampão de lise. </li><li> Centrifuga-se as amostras durante 5 min a 900 x g. Retire e elimine o sobrenadante. Verifique se o pellet é agora branco. NOTA: Se o pellet ainda aparece de vermelho, uma lise adicional pode ser necessária antes de prosseguir para a próxima etapa. </li><li> Re-suspender o sedimento em 1 ml de tampão de FACS. Centrifuga-se as amostras durante 5 min a 900 x g. Retire e elimine o sobrenadante. </li><li> Re-suspender o sedimento em 300 ul de tampão de FACS e quantificar as células GFP + utilizando citometria de fluxo (NOTA: Nesta altura desejada qualquer imunocoloração das células pode ser realizada, usando técnicas convencionais, antes da análise por citometria de fluxo). </li></ol>

<ol>
	<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.

Busulfano condicionado permite a geração de alto nível BM quimerismo em ratinhos com células hematopoiéticas, que são prontamente detectáveis. Este condicionamento pode ser realizado sem a necessidade de instalações de irradiação. Além disso, a mielossupressão com as doses de bussulfano descrito neste protocolo é bem tolerado, minimizando os efeitos secundários tóxicos causados ​​por doses mieloablativas de irradiação, e, portanto, pode ser uma técnica mais apropriada para gerar BM quimerismo em j...

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.

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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Busulfex</td>
			<td style="width:224px;">Otsuka Canada Pharmaceutical Inc.</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:224px;">Life Technologies</td>
			<td style="width:119px;">A10492-01</td>
			<td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:224px;">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.

A administração de 60-100 mg / kg de busulfano é geralmente bem tolerada pelos ratinhos. No entanto, os animais normalmente perdem peso durante a fase de condicionamento (~ 5-10%) e, assim, a dieta pode ter de ser complementada com guloseimas como irradiados sementes de girassol para promover a alimentação. Os níveis de quimerismo de células de&gt; 80% para GFP + no sangue periférico e BM deve ser consistentemente alcançável usando 60-100 mg / kg de busulfano 7. Nosso laboratório tem utilizado com ...

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.

Busulfan como Agente mielosupressores para a geração estável de alto nível de Medula Óssea quimerismo em Ratos

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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13.1K visualizações.  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.