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

The Wnt agonist R-spondin1 regulates systemic graft-versus-host disease by protecting intestinal stem cells

R-spondin1 stimulates the proliferation of intestinal stem cells through the Wnt signaling pathway and protects against graft-versus-host disease

Adult-specific systemic over-expression reveals novel in vivo effects of the soluble forms of ActRIIA, ActRIIB and BMPRII.

Bone morphogenetic proteins (BMPs)/growth differentiation factors (GDFs), which belong to the TGF-beta superfamily, are pleiotropic factors that play a role in regulating the embryonic development and postnatal homeostasis of various organs and tissues by controlling cellular differentiation, proliferation and apoptosis. Conventional transgenic and knockout (KO) mouse approaches have provided only limited information regarding the in vivo functions of BMP signaling in adult animals due to the effects on prenatal development and the difficulty in manipulating multiligand signals simultaneously. We recently produced transgenic chimeric mice(Tg chimeras) in which the soluble IgG1-Fc fusion protein of three BMP type II receptors (ActRIIA, ActRIIB, BMPRII) was highly circulated (281-709 μg/ml), specifically in adult mouse blood. Since each BMP receptor can bind to multiple BMP ligands, these Tg chimeras should be useful to investigate the effects of trapping multiple BMP ligands. Remarkably, some phenotypes were unexpected based on previous studies, such as KO mouse analyses, presumably representing the effects of the multiple ligand trapping. These phenotypes included increased red blood cells (RBCs) and decreased viability in adults. In a further study, we focused on the phenotype of increased RBCs and found that extramedullary hematopoiesis in the spleen, not in the bone marrow, was increased using histological and flow cytometric analyses. Although it remains to be elucidated whether the transgene products affect the tissues directly or indirectly, our data provide novel and important insight into the biological functions of the soluble IgG1-Fc fusion protein of three BMP type II receptors in adults, and our approach should have broad applications to research on other ligand receptor families and studies involving mouse models

Serum protein levels produced from the transgenes in each Tg chimeras.

<p>A. Serum hEPO concentrations of the four types of eight-week-old Tg chimeras identified using a combination of two different promoters (A: used in our previous report, B: newly developed for this study) and the existence of puro and neo selection markers. ***, P<0.001 (Student’s <i>t</i>-test). Significant differences were observed in both females and males. B. The serum concentration of each soluble protein (ActRIIA-Fc, ActRIIB-Fc and BMPRII-Fc) in 11- to 16-week-old Tg chimeras was measured using ELISA (see the Materials and Methods). In the control chimeras, the Fc protein was not detectable. As described in Materials and Methods, the “control” chimeras are produced using ES cells in which the expression unit is not introduced.</p

Summary and grouping of the phenotypes observed in each Tg chimeras (11- to 16 weeks old).

<p>A. Venn diagram representing the common phenotypes of the three Tg chimeras. The ellipses represent the phenotypes obtained from each Tg chimeras. The common phenotypes are shown in the overlapping sections. B. Summary of the phenotypes. The notes correspond to the data shown in Figure 3A. For example, VII means that the features were common to all three (ActRIIA-Fc, ActRIIB-Fc and BMPRII-Fc) Tg chimeras.</p

Increased extramedullary hematopoiesis was observed in the chimeric mouse spleens.

<p>Results of the flow cytometric analysis of the bone marrow (A) and spleen (B). The black bar indicates the control chimeras and the gray bar indicates the Tg chimeras. Samples were obtained from 8-week-old Tg chimeras (N=6). Each experiment was performed independently, and a control was prepared for each procedure. The ratio of [% of gated cells] in each Tg chimera to that observed in the control chimeras was described as [relative value]. *, P<0.05 ; **, P<0.01; †, tendency (0.05t-test).</p

Survival rate of each Tg chimeras.

<p>The term before 10 weeks was abbreviated because all chimeric mice were still alive. </p

Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency

Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research