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

Summary

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.

Abstract

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.

Protocol

1. Before-you-start Technical Considerations

1. 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.
2. 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).
3. Most mouse colonies are CD45.2. Our knockout mice belong to CD45.2 genotype (alternatively a CD45.2 WT mice source: Jackson Laboratories stock #000664). The determination of CD45.1 or....

Results

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&E staining of the colonic tissues. Colonic tissue structure changes and signs of inflammation can be quantitatively evaluated by H&E histology scoring system. Criteria for H&am.......

Discussion

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.......

Materials

List of materials used in this article

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