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RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development
J Chen1, R Lansford, V Stewart
1Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Harvard University Medical School, Boston.
Summary
This study introduces a novel RAG-2 deficient blastocyst complementation system to investigate gene function in lymphocyte development. The system successfully identified a critical role for immunoglobulin heavy chain genes in B cell maturation.
Area of Science:
- Immunology
- Developmental Biology
- Genetics
Background:
- Mature B and T lymphocytes are essential for adaptive immunity.
- Recombination-activating gene 2 (RAG-2) deficiency prevents VDJ recombination, leading to a lack of mature lymphocytes.
- Blastocyst complementation offers a method to study gene function in lymphocyte development.
Purpose of the Study:
- To establish and utilize a RAG-2 deficient blastocyst complementation system for evaluating gene function in lymphocyte differentiation and function.
- To investigate the role of immunoglobulin heavy chain joining (JH) gene segments in B cell development.
- To assess the rescue of B cell development by introducing a functional mu heavy-chain gene.
Main Methods:
- Utilized RAG-2 deficient mice and blastocysts.
- Generated somatic chimeras by injecting wild-type or genetically modified embryonic stem (ES) cells into RAG-2 deficient blastocysts.
- Employed ES cells with targeted mutations in JH gene segments (JH+/- and JH-/-).
- Assessed B and T cell development and function in the resulting chimeras.
- Transfected JH-/- ES cells with a functional mu heavy-chain gene to evaluate rescue of B cell development.
Main Results:
- RAG-2 deficient blastocyst complementation with normal ES cells generated chimeric animals with mature B and T cells.
- Complementation with JH+/- ES cells resulted in normal B and T cell development.
- Complementation with JH-/- ES cells led to normal T cell development but a complete block in B cell development.
- Transfection of a functional mu heavy-chain gene into JH-/- ES cells rescued B cell development, producing IgM-expressing B cells that responded to lipopolysaccharide stimulation.
Conclusions:
- The RAG-2 deficient blastocyst complementation system is effective for studying lymphocyte development and gene function.
- Immunoglobulin heavy chain joining gene segments are critical for early B cell development.
- Restoration of a functional mu heavy-chain gene can rescue the B cell developmental block caused by JH deficiency.