Name: differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation
Uploaded: 2012-10-01T12:00:00
Description: Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

This work was funded by Pilot and Feasibility Study grant from UCLA-CURE Center, the Crohn's and Colitis Foundation of America Career Development Award (#2691) and National Institute of Health NIDDK K01 (DK084256) funding to Hon Wai Koon.
Bone marrow irradiation operation was assisted by Bernard Levin and Scott Kitchen of UCLA Center for AIDS Research Mouse/Human Chimera Core facility. Flow cytometry operation was assisted by UCLA Vector Core facility.

1. Before-you-start Technical Considerations
<ol>
	<li>We recommend using both C57/BL6 wild-type mice and knockout mice for experiment because mice with corresponding CD molecules can be purchased from major mouse vendors.</li>
	<li>Since it is difficult to track the wild-type mice and knockout mice derived donor cells after bone marrow transplantation, it is necessary to use CD45.1 mice to represent wild-type mice. (CD45.1 C57/SJL WT mice Jackson Laboratories stock #002014).</li>
	<li>Most mouse colonies are CD45.2. O...

<ol>
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This bone marrow transplantation approach is suitable for immunology research of colitis, infection, cancer, obesity and other diseases. This bone marrow transplantation experiment is needed when the gene-of-interest is expressed in both bone marrow derived and non-bone marrow derived cells and the gene-of-interest is suspected to mediate disease by cells from either population. For example, antimicrobial peptide cathelicidin is shown to modulate acute colitis. But it is expressed in both epithelial cells and immune cell...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Cell staining buffer</td>
			<td>Biolegend</td>
			<td>#420201</td>
		</tr>
		<tr>
			<td>10X RBC lysis buffer</td>
			<td>Biolegend</td>
			<td>#420301</td>
		</tr>
		<tr>
			<td>FITC mouse isotype control</td>
			<td>Biolegend</td>
			<td>#400207</td>
		</tr>
		<tr>
			<td>PE mouse isotype control</td>
			<td>Biolegend</td>
			<td>#400211</td>
		</tr>
		<tr>
			<td>PE anti-mouse CD45.2</td>
			<td>Biolegend</td>
			<td>#109807</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.1</td>
			<td>Biolegend</td>
			<td>#110705</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32 blocking</td>
			<td>Biolegend</td>
			<td>#101301</td>
		</tr>
		<tr>
			<td>40 &mu;m cell strainer</td>
			<td>Fisherbrand</td>
			<td>#22363547</td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>MP biomedicals</td>
			<td>#1860454</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisherbrand</td>
			<td>#BP2425</td>
		</tr>
		<tr>
			<td>Sulfatrim (SMZ)</td>
			<td>Qualitest</td>
			<td>NC9242720 (fisher)</td>
		</tr>
		<tr>
			<td>Lysol IC</td>
			<td>Andwin Scientific</td>
			<td>NC9745686 (fisher)</td>
		</tr>
		<tr>
			<td>Vaccutainer</td>
			<td>BD</td>
			<td>#8000813</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Braintree Scientific</td>
			<td>#TV-150</td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>J.L. Shepherd and Associates</td>
			<td>Mark I 68A</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>BD FACSCanto II</td>
		</tr>
		<tr>
			<td>Flow cytometer test tube</td>
			<td>Falcon</td>
			<td>#352052</td>
		</tr>
		<tr>
			<td>Digital caliper</td>
			<td>Fisherbrand</td>
			<td>14-648-17</td>
		</tr>
		<tr>
			<td>Hemoccult ICT</td>
			<td>Beckman Coulter</td>
			<td>G0328QW</td>
		</tr>
	</tbody>
</table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

If the gene-of-interest plays a significant role in immune cells during development of colitis, the mice receiving bone marrow of different genotype through bone marrow transplantation (WT to KO or KO to WT) should have an altered response to DSS colitis. One of the most important parameters for determining the severity of colitis is the H&amp;E staining of the colonic tissues. Colonic tissue structure changes and signs of inflammation can be quantitatively evaluated by H&amp;E histology scoring system. Criteria for H&am...

Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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