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Reporter Gene Insertions Reveal a Strictly B Lymphoid-Specific Expression Pattern of Pax5 in Support of Its B Cell Identity Function¹

Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria

The transcription factor Pax5 is essential for B cell commitment and development. Although the detailed Pax5 expression pattern within the hemopoietic system is still largely unknown, we previously reported that Pax5 is monoallelically transcribed in pro-B and mature B cells. In this study, we have investigated the expression of Pax5 at single-cell resolution by inserting a GFP or human Cd2 indicator gene under the translational control of an internal ribosomal entry sequence into the 3' untranslated region of Pax5. These insertions were noninvasive, as B cell development was normal in Pax5^ihCd2/ihCd2 and Pax5^iGFP/iGFP mice. Transheterozygous Pax5^ihCd2/iGFP mice coexpressed GFP and human CD2 at similar levels from pro-B to mature B cells, thus demonstrating biallelic expression of Pax5 at all stages of B cell development. No reporter gene expression could be detected in plasma cells and non-B cells of the hemopoietic system. Moreover, the vast majority of common lymphoid progenitors and pre-pro-B cells in the bone marrow of Pax5^iGFP/iGFP mice did not yet express GFP, indicating that Pax5 expression is fully switched on only during the transition from uncommitted pre-pro-B cells to committed pro-B cells. Hence, the transcriptional initiation and B cell-specific expression of Pax5 is entirely consistent with its B cell lineage commitment function.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Boehringer Ingelheim and the Austrian Industrial Research Promotion Fund.

² Current address: Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, U.K.

³ Address correspondence and reprint requests to Dr. Meinrad Busslinger, Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. E-mail address: busslinger{at}imp.ac.at

⁴ Abbreviations used in this paper: MPP, multipotent progenitor; CLP, common lymphoid progenitor; IRES, internal ribosomal entry site; DN, double negative; LSK, Lin^–Sca1^highc-Kit^high progenitor.

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