Oberdörffer, Philipp (2003) Part I: B Cell Development and Function in Mice with a Targeted Lambda1 Light Chain Gene Insertion; Part II: Novel Approaches for Cre-mediated Inducible Gene Alteration in Mice. PhD thesis, Universität zu Köln.
Part I: The B cell antigen receptor (BCR) consists of two immunoglobulin heavy (IgH) chains, two immunoglobulin light (IgL) chains and the signaling molecules Ig alpha and Ig beta. The variable regions of IgH and IgL chains confer antigen specificity and are assembled from various gene segments through a unique series of somatic recombination events. Ig rearrangement is a developmentally regulated process with IgH rearranging prior to IgL. IgL rearrangements can take place on two distinct genomic loci, Ig lambda and Ig kappa, but occur more frequently at the latter. Previous results suggested that the unrearranged Ig kappa locus negatively regulates Ig lambda transcription and/or rearrangement. Here, I demonstrate that expression of a pre-rearranged lambda1 light chain gene inserted into the Ig lambda locus is independent of Ig kappa rearrangements. Expression of the inserted lambda1 gene is developmentally regulated like that of a pre-rearranged Ig kappa gene inserted into the Ig kappa locus and coincides developmentally with the occurrence of Ig kappa rearrangements in wild-type mice. I thus conclude that transcription of a gene rearrangement in the Ig lambda locus is developmentally controlled and not negatively regulated by the unrearranged Ig kappa locus. Mice carrying an inserted lambda1 light chain gene undergo secondary IgL rearrangements at a frequency that is comparable to WT mice. This process is presumably a consequence of receptor editing in B cells that expressed an auto-reactive IgH/Iglambda1 pair. Unlike Ig kappa rearrangements, Ig lambda rearrangements cannot be deleted from the genome and the resulting �receptor-edited� lambda1+ B cells express both the inserted lambda1 light chain and an endogenous Ig kappa molecule on the cell surface. While allelic and isotype exclusion generally ensure that B cells express a unique receptor specificity, my results indicate that dual receptor specificity poses no problem for the generation of mature B cells. However, B cells with two distinct receptor specificities are counterselected upon BCR-ligand interaction, which is a crucial event during immune responses and possibly in the generation of B1 cells. Counterselection of Ig kappa/Ig lambda double positive B cells can occur on the protein level and on the genomic level and ensures the generation of an antigen-specific immune response. Part II: For the last decade, Cre/loxP-mediated conditional mutagenesis has been instrumental to the study of gene function in mice. Both Cre-mediated deletion and inversion of loxP-flanked gene segments are widely used to manipulate the mouse genome. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not support the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that displays a favorable forward reaction equilibrium in vitro. Here, I demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate that has been targeted into the mouse genome. lox66/lox71-mediated recombination was two to three times less efficient than that of a WT loxP pair both in vitro and in vivo. Inversion efficiency was generally dependent on Cre expression levels and near complete lox66/lox71-mediated inversion could be observed in vivo using both an inducible and a cell-type specific cre transgene. Reverse recombination was undetectable in most tissues. Taken together, lox66/lox71 allows essentially unidirectional gene inversion. Its efficiency is, however, dependent on the expression level of the cre transgene of choice. Over the past few years, RNAi has been introduced as an additional means to manipulate gene expression. RNAi allows the simultaneous knock-down of several highly homologous gene products using a single short hairpin RNA (shRNA) encoding transgene. Here, I combined RNAi with Cre/loxP technology to create a system that will allow the cell type-specific and/or developmentally regulated induction of RNAi in mice. In order to control shRNA expression by Cre, I designed a loxP-flanked transcription termination cassette, which can be removed by Cre-mediated recombination. A transgene encoding this inducible expression vector has been integrated into the mouse HPRT locus. Efficient and sequence-specific knock-down of a target gene was observed with a single copy of this transgene in embryonic stem cells and this process was strictly dependent on Cre.
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