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Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation
Published on: October 1, 2012
Differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow
Hon Wai Koon1, Samantha Ho, Michelle Cheng
1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, The University of California Los Angeles, Los Angeles, CA, USA.
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.
Insights
This study uses bone marrow transplantation in mice to determine if a specific gene
Area of Science:
- Immunology
- Genetics
- Gastroenterology
Background:
- Understanding gene function in complex diseases like colitis requires dissecting cell-specific contributions.
- Bone marrow-derived cells play a critical role in immune responses and inflammation.
- Differentiating the roles of genes in hematopoietic versus non-hematopoietic cells is challenging.
Purpose of the Study:
- To investigate the specific role of a gene of interest in bone marrow-derived cells during colitis development.
- To establish a robust mouse model for studying cell-type-specific gene function in inflammatory diseases.
- To validate the use of bone marrow transplantation and CD markers for assessing experimental success.
Main Methods:
- Bone marrow transplantation was performed between wild-type and knockout mice, utilizing CD45.1 and CD45.2 markers to track donor/recipient cell populations.
- Colitis was induced using dextran sodium sulfate (DSS) in recipient mice post-bone marrow reconstitution.
- Flow cytometry was employed to quantify donor cell engraftment in blood and bone marrow, confirming successful transplantation.
Main Results:
- The study successfully generated chimeric mice with distinct bone marrow genotypes.
- The methodology allowed for the evaluation of gene function specifically in bone marrow-derived cells in the context of chemically induced colitis.
- Significant alterations in recipient mice phenotype post-transplantation indicate the gene's importance in bone marrow-derived cells for colitis development.
Conclusions:
- Bone marrow transplantation is an effective technique to study the role of specific genes in bone marrow-derived cells during disease.
- The success of bone marrow transplantation can be accurately assessed using CD markers and flow cytometry.
- This approach provides a powerful tool for dissecting the cellular mechanisms underlying inflammatory conditions like colitis.
