Azar, AA

Marshall-Clarke, S

Slupsky, J

Vlatkovic, N

The malignant cells of chronic lymphocytic leukemia (CLL) over-express PKCβII, a feature pathogenically important because the TCL1 mouse model of CLL fails to develop disease when PKCβ expression is disrupted. The purpose of this project was to generate transgenic mice in which PKCβII is over-expressed only in B cells. The central hypothesis of this thesis was that over-expression of PKCII in developing B cells will shift development to favour the generation of the B-1 and MZ B cell populations and eventually lead to the development of a CLL-like disease in the mouse. To construct the expression plasmid, an Eµ promoter was used to direct B cell-specific expression of PKCβII in transgenic mice (Eµ-PKCβII tg mice). PKCβII was tagged with haemagglutination antigen (HA) for identification in Western blots and immunohistochemistry (IHC). Also, mCherry was integrated within the expression plasmid construct in order to visualize transgenic B cells. Over-expressed PKCβII and mCherry fluorescence were detected when this plasmid was tested in A20 cells, a mouse B lymphoma cell line. The construct was then injected into pro-nuclei of fertilized ova isolated from pregnant mice. The injected ova were then transferred to recipient mice to generate potential founder mice. Sequential crossings of transgenic litters born from a single founder mouse led to the generation of homozygous Eµ-PKCβII tg mice. In the spleen of Eµ-PKCβII tg mice, the expression of the transgenic PKCIIHA was detected and quantification of PKCβII, using Western blotting, showed that PKCβII was over-expressed in Eµ-PKCβII tg mice compared with wild type mice. However, the mCherry could not be detected; this might be due to the fact that expression of the secondary gene(in this case mCherry) was not always efficient because of variable transcription from the IRES sequence. IHC analysis of the spleen of Eµ-PKCβII tg mice confirmed the expression of PKCIIHA in B cell-rich tissues where the staining for either HA or for PKCII showed the high expression of these proteins in the white pulp of the spleen specifically to the follicular region. There was no notable development of disease, CLL-like or otherwise in aged Eµ-PKCβII tg mice, but Flow cytometry analysis pointed out that the over-expression of PKCII resulted in a reduction in the proportion of follicular B cells and an increase in the proportion of MZ B cells in the spleen, and in B-1 cells in peritoneum and periphery of Eµ-PKCβII tg mice. This expansion of MZ B cells in the spleen was confirmed by H&E stains showing an enlarged marginal zone within the structure of the spleen, and IgM stains showed that this expanded MZ consists mainly of IgM+ cells. Thus, the generation of a mouse that over-expresses PKCII specifically within the B cell compartment led to an expansion in the populations of IgM+ B cells (MZ and B-1 B cells) and a reduction of follicular (mature) B cells. This mouse may be useful for the study of human CLL because of the potential to accelerate disease progression, and would therefore serve as a useful model for drug testing

Generation of PKCbII transgenic mice for the study of chronic lymphocytic leukaemia

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