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| Status |
Public on Jun 01, 2022 |
| Title |
Next Generation Sequencing Identifies Biased Light Chain Usage and Evidence of Light Chain Gene Replacement in phosphatidylcholine (PtC)-specific CD5+ B cells from dnRAG1 and VH12 mice. |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
B cells reactive toward phosphatidylcholine (PtC) are enriched in the B1 B cell subset, and express predominantly one of two VH/Vk combinations to confer this specificity: VH12/Vk4/5H and VH11/Vk9. Studies of transgenic mice expressing the VH12 heavy chain (VH12 mice) suggest two major checkpoints for light chain expression in this system: the first involves selection of V-J rearrangements which encode a “permissive” light chain that can functionally pair with the VH12 heavy chain; the second involves receptor editing to salvage non-PtC reactive B cells to acquire a permissive light chain that confers PtC reactivity. If this model is correct, impairing receptor editing should reduce the frequency of PtC-reactive B1 B cells in VH12 mice. To test this possibility, we bred VH12 mice to transgenic mice expressing a catalytically inactive form of RAG1 (dnRAG1 mice) which show a defect in receptor editing. Interestingly, dnRAG1 expression in VH12 mice enforces development of PtC-reactive B1 B cells, rescuing the loss of splenic B cells observed in VH12 mice. These data suggest receptor editing normally functions to remove a large portion of PtC-specific B cells in VH12 mice. Deep sequencing of the expressed light chain repertoire of PtC-reactive and non-reactive B cells in VH12 mice revealed that PtC-reactive B cells predominantly expressed the Vk4/5H (IGKV4-91) light chain gene, whereas PtC-non-reactive B cells expressed a broader, yet restricted, set of light chain genes. This analysis also revealed a low frequency of in-frame hybrid light chain genes appearing to originate via Type 2 gene replacement, which we show can originate from template switching during PCR.
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| Overall design |
The sequencing libraries were generated from total RNA extracted from 60 samples. 24 samples were analyzed in the first round, these were comprised of 3 independent biological replicates for 8 unqiue cell populations from the spleens of 4 geneotypes of mice. For the second round 36 samples were anlysed, comprised of 3 independent biological replicates for 12 unique cell populations from the bone marrow and spleen of 2 genotypes of mice.
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| Contributor(s) |
Worth AN, Palmer VL, Schabla NM, Perry GA, Fraser-Philbin AN, Swanson PC |
| Citation(s) |
35705027 |
| |
| Submission date |
Jan 29, 2021 |
| Last update date |
Aug 31, 2022 |
| Contact name |
Patrick Swanson |
| Organization name |
Creighton University School of Medicine
|
| Department |
Medical Microbiology and Immunology
|
| Lab |
Swanson
|
| Street address |
2500 California Plaza
|
| City |
Omaha |
| State/province |
NE |
| ZIP/Postal code |
68178 |
| Country |
USA |
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| Platforms (1) |
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| Samples (60)
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| Relations |
| BioProject |
PRJNA697869 |
| SRA |
SRP303817 |
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