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Chapters
- 00:05Title
- 01:24Splenic B-Lymphocyte Isolation and Stimulation
- 05:42Transfection of Producer Cells
- 07:26Infection of Stimulated Splenic B cells
- 08:12Flow Cytometry Analysis
- 08:40Rescue of AID-Deficiency by Transduction of AID-Deficient B Cells with a Recombinant Retrovirus
- 08:57Conclusion
An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.
Tags
Recombinant Retroviral ProductionB CellsTransgenic ExpressionReverse Genetic ApproachHeterologous Expression SystemPhenotype AnalysisGene Over-expressionHematopoetic SystemMouse GeneticsHuman Genes And DiseasesSynteny ConservationGene DisruptionsAllelic ReplacementTransgenic Mouse CreationCell Culture Model
