Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells.

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.

Identification of the renal erythropoietin-producing cells using transgenic mice.

Regulation of erythropoietin production by the kidneys is central to the control of erythropoiesis. Uncertainty about the identity of the renal cells involved has been a major obstacle to understanding this mechanism. We have used sequence from the mouse erythropoietin locus to direct expression of a marker gene, SV40 T antigen, to these cells in transgenic mice. The transgenic constructs contained an oligonucleotide marker (Epo-M) or SV40 sequence (Epo-TAg) in the 5' untranslated region of the mouse erythropoietin gene, flanked on each side by 9 and 7.5 kb of DNA from the mouse erythropoietin locus. Anemia-inducible expression of Epo-M and Epo-TAg was observed in the kidney. In one of thirteen lines, homologous integration of Epo-TAg into the mouse erythropoietin locus occurred. In transgenic mice bearing Epo-TAg at homologous and heterologous insertion sites, renal expression was restricted to a population of cells in the interstitium of the cortex and outer medulla. Immunohistochemical characterization by light and electron microscopy shows that these are the fibroblast-like type I interstitial cells.

The influence of handling procedures during mouse oocyte and embryo recovery on viability and subsequent development in vitro.

The effect on mouse oocyte and embryo viability of prolonging the time that they spend in the oviduct prior to flushing has been investigated. Oocytes and embryos from oviducts that were flushed quickly and with as little temperature variation as possible were compared with oocytes and embryos flushed from oviducts that had been left for 30 min, either at 37 degrees C in saline or in the intact dead mouse before dissection. It was found that retention of the eggs and embryos in the oviducts leads to a lower viability at recovery and also affects the subsequent developmental potential of embryos in culture.