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.

注：伦理学声明：此协议已审查和批准由西蒙·弗雷泽大学的动物护理委员会（UACC;允许数字1037K-12和1060K-03），并符合加拿大议会动物保育，美国国立卫生研究院指南护理和使用实验动物，以及EEC理事会指令。 1.特别注意事项注：白消安是细胞毒性。处理并根据材料安全数据表和制度规定进行处置。 <ol><li>执行所有的技术在无菌层流罩。 </li><li>消毒通过包装在合适的包装材料，诸如剥离封装，并高压灭菌，使用前的仪器。 </li><li>而相比于照射为感染的风险是低带马利兰调节，仅在层流罩的前2周后移植处理的动物。 </li></ol> 2.调理受体小鼠<ol><li>稀马利兰至3毫克/毫升，用无菌磷酸盐缓冲盐水（PBS）中。 注意：为了让新鲜稀释至白消安注射液使用的每一天之前是很重要的。 </li><li>日常管理20毫克/公斤的白消安稀释到受体小鼠通过IP注射。 </li><li>重复的20毫克/千克马利兰每日IP注射，直到60-100毫克总剂量/千克已经交付（ 即，对于一个80毫克/公斤的总剂量，给药白消安，连续4天的20毫克/千克）。 </li></ol> 3.隔离供体骨髓细胞的注：此协议已被成功地用于从最多5供体小鼠中分离和制备BM细胞。通常每只小鼠的细胞产量是约30-40百万DC培养上清，这足以移植12-16受体小鼠。如果需要更多的供体小鼠中的协议可能需要作相应调整。 <ol><li>继白消安空调euth的最后一天anize使用的CO 2与GFP供体小鼠（1至6个月大）（或通过在接纳机构其他安乐死过程）。为了避免移植并发症使用同基因供体是同性的收件人。 </li><li>喷小鼠用70％的乙醇。升降机皮肤在腹部，并使用外科剪刀通过从腹腔向上朝向脚踝腿部皮肤做一个切口。握住脚，坚决从拉向臀部露出腿部组织脚踝的皮肤。 </li><li>修剪掉肌肉和脂肪组织从股骨以暴露髋关节。 </li><li>同时轻轻拉动脚伸腿，按压髋关节的剪刀。剪刚股骨，注意不要切股骨本身的头上。 </li><li>为了帮助保持无菌，由脚抱腿和摩擦骨表面灭菌组织清理从骨子里任何剩余的组织。 </li><li>通过在膝关节切削分离股骨和胫骨和放置在含有无菌PBS培养皿股骨。在冰上孵育。 </li><li>取出并削减在那里的腓骨连接到胫骨点丢弃腓骨。将胫骨在培养皿与股骨和在冰上孵育。 </li><li>对于另一条腿重复步骤3.2-3.7，并在必要时，附加的供体小鼠。下面除去的骨头，消毒的外科手术工具用热珠灭菌或使用一组新的无菌工具用于随后的步骤。 </li><li>对于股骨，抱钳股骨，用手术剪刀仔细地&#39;刮&#39;的远端脱骨。去除尽可能少，以暴露骨髓腔​​必要的骨。 </li><li>填充用3毫升无菌PBS的注射器，并附加一个23政针。仔细孔针插入BM腔冲洗BM到无菌培养皿。一定要凑髓腔用针点，以保证除去所有需要的细胞。以下提取，确保红色BM不再可见和骨现在显示为白色。 </li><li>随后的股骨重复步骤3.9-3.10，汇集所有的BM在同一培养皿。 </li><li>为胫骨，保持胫骨钳，并仔细&#39;剃须&#39;，其中所述胫骨附着到膝盖以暴露骨髓腔​​的末端。使沿其中可见红色BM结束骨第二晋级。 </li><li>填用3ml无菌PBS的注射器，并附加一个地下25针。轻轻插入针头插入骨髓腔（从附着到膝盖该端），并刷新BM到从股骨含有BM培养皿。刮去骨髓腔的壁与针以除去所有的细胞。以下提取，确保红色BM不再可见和骨现在显示为白色。 </li><li>随后的胫骨，重复步骤3.12-3.13，汇集所有的BM在同一个培养皿。 </li><li>轻轻磨碎的BM与1毫升枪头分离的CELLS。 </li><li>通过40μM过滤器篮的细胞悬浮到无菌50mL离心管中。冲洗用PBS菜获取任何剩余的细胞，并通过过滤器转移到这一点的离心管中。 </li><li>离心5分钟，在450×g离心在4℃下。取出并弃去上清液。 </li><li>重悬在3ml红细胞裂解缓冲液沉淀。在冰上孵育8.5分钟。 </li><li>加约30毫升无菌PBS中以猝灭裂解缓冲液中。 </li><li>离心5分钟，在450×g离心在4℃下。取出并弃去上清液。 </li><li>重新悬浮于1ml无菌PBS的沉淀。保持在冰上。 </li><li>取一小等分试样的细胞匀浆和稀释用PBS（1：100 1小鼠; 1：200为2小鼠等）使用血细胞计数器来量化细胞数。 </li><li>稀释细胞悬液，以8×10 6个细胞/ ml，并在冰上孵育直至注射。 </li></ol> 4.骨髓移植<ol><li>网络连接LL 0.5ml注射器（用28克胰岛素针）用300微升稀释的细胞悬浮液对每个小鼠进行注射。一定要消除存在于注射器任何气泡。 </li><li>上放置装有一个加热垫的空调受体小鼠鼠笼并允许尾静脉扩张。 </li><li>就拿收件人鼠标和地方约束装置。擦拭用外科纱布浸透70％乙醇的尾部。 </li><li>牢固地保持在尾部，并与斜面向上，轻轻插入针头插入的侧面尾静脉中的一个，并注入该细胞悬浮液。 </li><li>保持手术纱布在注射部位，直到前引入鼠标到一个新的干净的笼子出血停止。 </li></ol> 5.估计BM嵌合体的程度注：嵌合体的水平是在外周血典型变量1周后的骨髓移植，因此嵌合体的水平，通常估计在至少2周后的骨髓移植。詹水平erism应达到&gt; 80％的GFP +细胞在外周血3-4周内后骨髓移植。 <ol><li>制备FACS缓冲液（2mM EDTA的+ 2％胎牛血清的PBS血清）。 注：FACS缓冲液可以储存在4℃几周。 </li><li>鼠标放置在一个约束管刮胡子的皮毛上的小腿后侧暴露侧隐静脉。 </li><li>薄外套凡士林和皮尔斯隐静脉将25g针头的腿。 </li><li>收集大约50微升血液与肝素涂层的毛细管。加血液至含有1ml的FACS缓冲液中的微离心管中。混合管通过倒置并储存在冰上。 </li><li>按住注射部位的手术纱布，直到出血停止返回鼠标回到笼子前。 </li><li>离心血液样品5分钟，在900×g下。取出并弃去上清液。 </li><li>重悬在500μl的红细胞裂解缓冲液沉淀。在冰上孵育8.5分钟。 </li><li>加入1毫升的FACS缓冲液，淬灭裂解缓冲液中。 </li><li>离心样品5分钟，在900×g下。取出并弃去上清液。 </li><li>执行在500μl的红细胞裂解缓冲液的第二裂解步骤。在冰上孵育8.5分钟。 </li><li>加入1毫升的FACS缓冲液，淬灭裂解缓冲液中。 </li><li>离心样品5分钟，在900×g下。取出并弃去上清液。检查球现在是白色的。 注意：如果该粒料仍然出现红色，附加的裂解步骤可能需要在进行下一步骤之前。 </li><li>重新悬浮于1ml FACS缓冲液沉淀。离心样品5分钟，在900×g下。取出并弃去上清液。 </li><li>重悬在300微升FACS缓冲液将沉淀，并使用流式细胞术定量的GFP +细胞（注意：在这一点上可进行该细胞的任何期望的免疫染色，使用标准技术，前流式细胞仪分析）。 </li></ol>

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The authors have nothing to disclose.

马利兰调理允许高层骨髓嵌合小鼠造血细胞是容易检测的产生。可以无需照射设施来执行此调节。此外，骨髓抑制与剂量在这个协议中概述马利兰的耐受性良好，最小化毒副作用引起的清髓性剂量的辐射，并因此可能是一个更合适的技术，以产生BM嵌合在年轻的，年老，或患病的小鼠更容易清髓性照射。一些RIC协议使用低的，非myleoablative剂量全身照射，以尽量减少不良副作用，但这些协议仍然需?...

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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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:224px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</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>
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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毫克/公斤白消安是普遍受到老鼠的耐受性。然而，动物通常在调节阶段（〜5-10％），因此饮食减肥可能需要与对待诸如照射葵花籽鼓励饮食进行补充。 &gt; 80％的GFP +细胞在外周血嵌合和BM的水平应始终实现使用60-100毫克/公斤马利兰7。我们的实验室已成功地利用这个协议产生超过100嵌合体小鼠&gt; 80％GFP +细胞在外周血和骨髓，几乎我们所有的BMTs使用同基因捐献者和接受者的时候是?...

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.

白消安作为骨髓抑制剂在小鼠生成稳定的高水平骨髓嵌合体

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 次观看.  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.