double-replacement gene targeting in mouse embryonic stem cells - creation of a mouse model for congenital polycythemia

Academic Article

Abstract

  • Erythropoietin (EPO) is the main regulator of erythroid survival, proliferation and differentiation and acts on the progenitor cells through its specific receptor - erythropoietin receptor (EPOR). Mutations of EPOR are known to be involved in pathogenesis of primary familial and congenital polycythemia (PFCP). We used double-replacement ("tag and exchange") gene targeting in murine embryonic stem (ES) cells to replace a mouse EPOR gene with its human (wild type and mutant) homolog. In the first step, the mouse EPOR gene was deleted in ES cells via homologous recombination and the locus was tagged with a positively selectable marker - hypoxanthine phosphoribosyl-transferase (HPRT) minigene. The correctly targeted ES cell lines were tested in vivo for their totipotency. The EPOR knockout (KO) phenotype was characterized in vivo and in vitro using both the yolk sac and fetal liver progenitors and progenitors from EPOR KO ES cell derived embryoid bodies. Cell lines that transmitted the KO allele to the germline were used for the "exchange" step - a second gene targeting event. The exchange vector contained the wild type and mutant human EPOR gene flanked by 5' and 3' mouse EPOR homology sequences. Negative selection (6thioguanine) against HPRT was used to select for homologous recombinants which lost the HPRT minigene through replacement by human EPOR gene. The animals with the wild type human EPOR are being analyzed. To assess the role of EPOR gene expression in erythroid and nonerythroid tissues, we will test whether erythroid specific expression of EPOR rescues the EPOR knockout. Double replacement gene targeting allows the creation of an animal model of PFCP. This strategy will be used to test specific mutations which lead to abnormal EPO/EPOR signaling. Mutagenized or chimeric EPOR gene constructs will be used to evaluate a role of EPO/EPOR pathways in erythroid commitment, proliferation, differentiation and maturation.
  • Published In

    Author List

  • Divoky V; Ryan T; Townes TM; Prchal JT
  • Start Page

  • 765
  • Volume

  • 26
  • Issue

  • 8