माउस बृहदांत्रशोथ में इम्यून और गैर प्रतिरक्षा कोशिकाओं से अस्थि मज्जा प्रत्यारोपण द्वारा जीन एक्सप्रेशन के कार्यात्मक भूमिका फर्क
Summary
अस्थि मज्जा प्रत्यारोपण अस्थि मज्जा व्युत्पन्न कोशिकाओं के जीनोटाइप को बदलने के लिए एक रास्ता प्रदान करता है. अगर ब्याज की जीन दोनों अस्थि मज्जा व्युत्पन्न कोशिकाओं और गैर अस्थि मज्जा व्युत्पन्न कोशिकाओं में व्यक्त किया है, अस्थि मज्जा प्रत्यारोपण अस्थि मज्जा व्युत्पन्न कोशिकाओं गैर अस्थि मज्जा व्युत्पन्न सेल जीनोटाइप बदलने के बिना एक अलग जीनोटाइप बदल सकते हैं.
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
Representative Results
Discussion
यह अस्थि मज्जा प्रत्यारोपण दृष्टिकोण इम्यूनोलॉजी बृहदांत्रशोथ के अनुसंधान, संक्रमण, कैंसर, मोटापा और अन्य बीमारियों के लिए उपयुक्त है. यह अस्थि मज्जा प्रत्यारोपण के प्रयोग की जरूरत है जब जीन की ब्या?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम पायलट और केंद्र UCLA इलाज Crohn है और बृहदांत्रशोथ फाउंडेशन अमेरिका कैरियर विकास पुरस्कार (2691 #) और स्वास्थ्य NIDDK K01 के राष्ट्रीय संस्थान (DK084256) के लिए धन माननीय वाई Koon से व्यवहार्यता अध्ययन अनुदान द्वारा वित्त पोषित किया गया था.
अस्थि मज्जा विकिरण आपरेशन बर्नार्ड लेविन और एड्स अनुसंधान माउस / मानव कल्पना कोर सुविधा के लिए UCLA केंद्र के स्कॉट रसोई द्वारा सहायता प्रदान की थी. प्रवाह cytometry आपरेशन UCLA वेक्टर कोर सुविधा द्वारा सहायता प्रदान की थी.
Materials
| Name of the reagent | Company | Catalogue number |
| 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 |
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