A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras

Total RNA samples prepared from 10 different tissues of 4-week-old hEPO/ΔRS and control ΔRS chimaeras were subjected to RT–PCR analysis using hEPO (35 cycles) and Cκ (35 cycles) specific primer pairs

<p><b>Copyright information:</b></p><p>Taken from "A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins"</p><p>Nucleic Acids Research 2005;33(9):e85-e85.</p><p>Published online 24 May 2005</p><p>PMCID:PMC1140086.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The sizes of resulting PCR products in all experiments were consistent with the expected sizes predicted from reported cDNA sequences. Murine GAPDH (25 cycles) was used as a control