Rescue of Erythropoietin-Deficient Mice From Anemia by Complementation with BAC Transgene.

Abstract 3614

Poster Board III-550

To maintain tissue oxygen homeostasis, erythropoietin (Epo) regulates the erythropoiesis. During embryonic development Epo is produced in the liver, but after birth and throughout adulthood Epo is produced and secreted from the kidney and support definitive erythropoiesis in the bone marrow. Anemia or hypoxic stress provokes transcription of the Epo gene and the mechanisms how the Epo gene expression is activated in a tissue-specific and hypoxia-inducible manner are one of the research topics of the molecular biology or gene regulation study. Since Epo-null mice die in utero of definitive erythropoiesis failure in the fetal liver, physiological significance of the Epo production for the adult erythropoiesis remains to be clarified. We have found that transgenic reporter gene expression driven by a BAC clone containing 180-kb mouse Epo gene locus recapitulates the Epo gene expression in the liver and kidney. To examine whether the Epo gene transcriptional activity within the 180-kb BAC is sufficient to recapitulate physiological Epo production, we attempted to rescue Epo-null mice from embryonic lethality with a BAC transgene harboring a floxed allele of the Epo gene. We found that transgene-derived Epo recovered the erythropoiesis in the fetal liver of Epo-null mice and Epo-null mice with the BAC transgene were born showing a Mendelian ratio. The rescued mice showed no hematological abnormality and were fertile. In the rescued mice bleeding anemia transiently increased plasma Epo level and after recovery from anemia plasma Epo level declined similarly to wild-type mice. In an RT-PCR analysis Epo production in the liver and kidney of the rescued mice was practically equivalent to that in the wild-type counterpart, while other tissues produced only marginal amount of Epo. These results thus demonstrate that the BAC transgene contains the Epo gene regulatory domain region sufficient to fully recapitulate the endogenous Epo gene expression and to compensate for the Epo-deficiency. We also found that the Epo gene deletion from the BAC transgene by general deletor-Cre transgene provoked the Epo-null phenotype, demonstrating that conditional inactivation of the Epo gene is attained in the BAC-rescued mice. The latter system will be a precious tool to investigate adult stage-specific roles of Epo.