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.

NOTE: Ethics Statement: This protocol has been reviewed and approved by the Animal Care Committee of Simon Fraser University (UACC; permit numbers 1037K-12 and 1060K-03) and is in compliance with the Canadian Council on Animal Care, the NIH Guide for the Care and Use of Laboratory Animals, and the EEC Council Directive.
1. Special Considerations
NOTE: Busulfan is cytotoxic. Handle and dispose according to the material safety data sheet and institutional guidelines.
<ol>
	<li>Perform all techniques aseptically in a laminar flow hood.</li>
	<li>Sterilize instruments prior to use by wrapping in an appropriate packaging material, such as a peel package, and autoclaving.</li>
	<li>While the risk for infection is lower with busulfan conditioning compared to irradiation, handle animals only in a laminar flow hood for the first 2 weeks following transplantation.</li>
</ol>
2. Conditioning of Recipient Mice
<ol>
	<li>Dilute busulfan to 3 mg/ml with sterile phosphate buffered saline (PBS). 
	NOTE: It is important to make a fresh dilution of busulfan just prior to use each day of injection.</li>
	<li>Administer 20 mg/kg of diluted busulfan to recipient mice via IP injection daily.</li>
	<li>Repeat daily IP injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been delivered (i.e., for an 80 mg/kg total dose, administer 20 mg/kg of busulfan for 4 consecutive days).</li>
</ol>
3. Isolation of Donor Bone Marrow Cells
NOTE: This protocol has been successfully used for isolating and preparing BM cells from up to 5 donor mice. Typically the cell yield per mouse is approximately 30-40 million BMDCs, which is sufficient to transplant 12-16 recipient mice. If more donor mice are needed the protocol may need to be adjusted accordingly.
<ol>
	<li>Following the last day of busulfan conditioning euthanize a GFP donor mouse (one to six months old) using CO2 (or by other euthanasia procedure accepted at institution). To avoid graft complications use syngeneic donors that are the same sex as the recipients.</li>
	<li>Spray mouse with 70% ethanol. Lift skin at the abdomen and using surgical scissors make an incision through the skin from the abdominal cavity up the leg towards the ankle. Holding the foot, firmly pull the skin from the ankle towards the hip exposing the leg tissue.</li>
	<li>Trim away muscle and fat tissue from the femur to expose the hip joint.</li>
	<li>While gently pulling on the foot to extend the leg, press the scissors against the hip joint. Cut just above the head of the femur taking care not to cut the femur itself.</li>
	<li>To help maintain sterility, hold the leg by the foot and clean any remaining tissue from the bones by rubbing the bone surface with autoclaved tissues.</li>
	<li>Separate the femur and tibia by cutting through the knee joint and place the femur in a culture dish containing sterile PBS. Incubate on ice.</li>
	<li>Remove and discard the fibula by cutting at the points where the fibula connects to the tibia. Place the tibia in the culture dish with the femur and incubate on ice.</li>
	<li>Repeat steps 3.2-3.7 for the other leg, and if necessary, additional donor mice. Following removal of the bones, sterilize the surgical tools with a hot bead sterilizer&#160;or use a new set of sterile tools for the subsequent steps.</li>
	<li>For the femurs, hold the femur with forceps and using surgical scissors carefully &#8216;shave&#8217; the distal ends off the bone. Remove as little of the bone as necessary to expose the BM cavity.</li>
	<li>Fill a syringe with 3 ml of sterile PBS and attach a 23 G&#160;needle. Carefully bore the needle into the BM cavity and flush the BM into a sterile culture dish. Be sure to scrape the medullary cavity with the needle point to ensure removal of all desired cells. Following extraction, ensure that the red BM is no longer visible and the bone now appears white.</li>
	<li>Repeat steps 3.9-3.10 for subsequent femurs, pooling all of the BM in the same culture dish.</li>
	<li>For the tibiae, hold the tibia with forceps and carefully &#8216;shave&#8217; the end where the tibia was attached to the knee to expose the BM cavity. Make a second cut along the bone where the visible red BM ends.</li>
	<li>Fill a syringe with 3 ml of sterile PBS and attach a 25 G&#160;needle. Gently insert the needle into the BM cavity (from the end that was attached to the knee) and flush the BM into the culture dish containing the BM from the femurs. Scrape the walls of the BM cavity with the needle to remove all cells. Following extraction, ensure that the red BM is no longer visible and the bone now appears white.</li>
	<li>Repeat steps 3.12-3.13 for subsequent tibiae, pooling all of the BM in the same culture dish.</li>
	<li>Gently triturate the BM with a 1 ml pipette tip to dissociate the cells.</li>
	<li>Pass the cell suspension through a 40 &#956;M basket filter into a sterile 50 mL centrifuge tube. Rinse the dish with PBS to get any remaining cells and transfer this through the filter into the centrifuge tube.</li>
	<li>Centrifuge for 5 min at 450 x g at 4 &#176;C. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 3 ml of erythrocyte lysing buffer. Incubate on ice for 8.5 min.</li>
	<li>Add ~30 ml of sterile PBS to quench the lysing buffer.</li>
	<li>Centrifuge for 5 min at 450 x g at 4 &#176;C. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 1 ml of sterile PBS. Keep on ice.</li>
	<li>Take a small aliquot of the cell homogenate and dilute with PBS (1:100 for 1 mouse; 1:200 for 2 mice, etc.) to quantify the cell number using a haemocytometer.</li>
	<li>Dilute the cell suspension to 8 x 106 cells/ml and incubate on ice until injection.</li>
</ol>
4. Bone Marrow Transplantation
<ol>
	<li>Fill a 0.5 ml syringe (with a 28 G&#160;insulin needle) with 300 &#956;l of diluted cell suspension for each mouse to be injected. Be sure to remove any air bubbles present in the syringe.</li>
	<li>Place the mouse cage containing the conditioned recipient mice on a heating pad and allow the tail veins to dilate.</li>
	<li>Take a recipient mouse and place in the restraining device. Wipe the tail with surgical gauze soaked with 70% ethanol.</li>
	<li>Firmly hold the tail and with the bevel up, gently insert the needle into one of the lateral tail veins and inject the cell suspension.</li>
	<li>Hold surgical gauze on the injection site until bleeding stops prior to introducing the mouse into a new clean cage.</li>
</ol>
5. Estimating the Extent of BM Chimerism
NOTE: Chimerism levels are typically variable in the peripheral blood at 1 week post-BMT, and as such chimerism levels are normally estimated at least 2 weeks post-BMT. Levels of chimerism should reach &#62;80% GFP+ cells in the peripheral blood within 3-4 weeks post-BMT.
<ol>
	<li>Prepare FACS buffer (2 mM EDTA + 2% fetal bovine serum in PBS). 
	NOTE: FACS buffer can be stored at 4 &#176;C for several weeks.</li>
	<li>Place the mouse in a restraining tube and shave the fur on the posterior leg to expose the lateral saphenous vein.</li>
	<li>Thinly coat the leg with petroleum jelly and pierce the saphenous vein with a 25 G&#160;needle.</li>
	<li>Collect approximately 50 &#956;l of blood with a heparin coated capillary tube. Add the blood to a micro-centrifuge tube containing 1 ml of FACS buffer. Mix the tube by inversion and store on ice.</li>
	<li>Hold surgical gauze on the injection site until bleeding stops before returning mouse back to the cage.</li>
	<li>Centrifuge the blood samples for 5 min at 900 x g. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 500 &#956;l of erythrocyte lysing buffer. Incubate on ice for 8.5 min.</li>
	<li>Add 1 ml of FACS buffer to quench the lysing buffer.</li>
	<li>Centrifuge the samples for 5 min at 900 x g. Remove and discard the supernatant.</li>
	<li>Perform a second lysis step in 500 &#956;l of erythrocyte lysing buffer. Incubate on ice for 8.5 min.</li>
	<li>Add 1 ml of FACS buffer to quench the lysing buffer.</li>
	<li>Centrifuge the samples for 5 min at 900 x g. Remove and discard the supernatant. Check that the pellet is now white. 
	NOTE: If the pellet still appears red, an additional lysis step may be needed before proceeding to the next step.</li>
	<li>Re-suspend the pellet in 1 ml of FACS buffer. Centrifuge the samples for 5 min at 900 x g. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 300 &#956;l of FACS buffer and quantify GFP+ cells using flow cytometry (NOTE: At this point any desired immunostaining of the cells can be performed, using standard techniques, prior to flow cytometric analysis).</li>
</ol>

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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. -. 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The authors have nothing to disclose.

Busulfan conditioning allows for the generation of high-level BM chimerism in mice with hematopoietic cells that are readily detectable. This conditioning can be performed without the need for irradiation facilities. Moreover, myelosuppression with the doses of busulfan outlined in this protocol is well tolerated, minimizing toxic side effects caused by myeloablative doses of irradiation, and thus may be a more suitable technique to generate BM chimerism in young, old, or diseased mice that are more susceptible to myeloa...

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 border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="21">
			<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>
	</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.

The administration of 60-100 mg/kg of busulfan is generally well tolerated by mice. However, animals typically lose weight during the conditioning phase (~5-10%) and thus the diet may need to be supplemented with treats such as irradiated sunflower seeds to encourage eating. Levels of chimerism of &#62;80% GFP+ cells in the peripheral blood and BM should be consistently achievable using 60-100 mg/kg busulfan7. Our laboratory has successfully used this protocol to generate over 100 chimeric mice with &#62;80% G...

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.

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 Views.  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.