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Analysis of C-MYC function in normal cells via conditional gene-targeted mutation
I M de Alboran1, R C O'Hagan, F Gärtner
1Howard Hughes Medical Institute and Children's, Hospital, Center for Blood Research and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.
Insights
Conditional c-myc gene inactivation in mice revealed its critical role in cell proliferation. c-Myc deficiency severely impaired proliferation in both mouse embryonic fibroblasts and B lymphocytes.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- Germline inactivation of the c-myc proto-oncogene in mice leads to embryonic lethality, highlighting its essential role.
- Understanding c-myc's function in specific cell types requires conditional gene manipulation strategies.
Purpose of the Study:
- To investigate the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes using a conditional knockout approach.
- To elucidate the impact of c-myc deficiency on cell proliferation and activation markers in these cell types.
Main Methods:
- Utilized a LoxP/Cre-based conditional mutation system for targeted c-myc gene inactivation in mice.
- Assessed proliferation rates of wild-type and c-Myc-deficient MEFs following Cre expression.
- Analyzed proliferation and activation marker expression (including CD95 and CD95 ligand) in wild-type and c-Myc-deficient B cells stimulated with anti-CD40 plus IL-4.
Main Results:
- Cre-mediated c-myc deletion reduced proliferation in wild-type MEFs, with a more pronounced effect in c-Myc-deficient MEFs.
- While Cre expression did not affect wild-type B cell proliferation, c-Myc deficiency severely impaired proliferation in response to anti-CD40 plus IL-4.
- c-Myc-deficient B cells upregulated early activation markers but not CD95 or CD95 ligand.
Conclusions:
- c-Myc plays a significant role in regulating proliferation in both fibroblasts and B lymphocytes.
- The findings suggest context-dependent functions of c-myc in cell activation and survival pathways.
- Potential limitations of the Cre-mediated gene inactivation approach were considered in the interpretation of results.