TECHNOLOGY REPORT Generation of a Conditional Knockout Allele for the Janus Kinase 2 (Jak2) Gene in Mice Andrea Krempler, 1 Yongyue Qi, 1 Aleata A. Triplett, 1 Jianqiong Zhu, 2 Hallgeir Rui, 2 and Kay-Uwe Wagner 1 * 1 Eppley Institute for Research in Cancer and Allied Diseases and the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 2 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC Received 13 April 2004; Accepted 4 June 2004 Summary: To study biologically relevant functions of the Janus kinase 2 (Jak2) in multiple cytokine and hormone receptor signal transduction pathways, we generated a conditional knockout (floxed) allele of this gene by plac- ing loxP sites around the first coding exon of Jak2. Ho- mozygous floxed animals developed normally and ex- hibited no phenotypic abnormalities. The conversion of the floxed allele into a null mutation was achieved by transmitting the targeted allele through the female germline of MMTV-Cre (line A) mice. Embryos that carry two Jak2 null alleles died around midgestation and ex- hibited impaired definitive erythropoiesis, which is a hallmark of Jak2 deficiency reported previously in con- ventional knockouts. This observation suggested that the Cre-mediated deletion of the first coding exon re- sults in a true null mutation that is incapable of mediat- ing signals through the erythropoietin receptor. Using mouse embryonic fibroblasts derived from Jak2 null em- bryos and their wildtype littermate controls, we dem- onstrated that Jak2-deficiency decouples growth hormone-receptor signaling from its downstream me- diators, the signal transducer and activator of tran- scription (Stat) 5a and 5b. genesis 40:52–57, 2004. © 2004 Wiley-Liss, Inc. Key words: Janus kinase 2; Stat5; embryonic development; Cre recombinase; gene targeting Jak-Stat pathways mediate extracellular polypeptide sig- nals, such as hormones and cytokines, from transmem- brane receptors to target gene promoters in the nucleus. Consequently, cells respond to these signals by altering their gene expression and, in turn, change their growth properties and their physiological activities (Aaronson et al., 2002). Four members of the Janus kinase (Jak) family have been identified in mammals: Jak1, Jak2, Tyk2, and Jak3. Genetic studies in mice revealed that the biological functions of individual Janus kinases are pleiotropic, which is consistent with their suggested coupling to multiple cytokine receptors in specific target cells cre- viewed by Kisseleva et al., 2002). In particular, Jak2 is suggested to mediate signals through single-chain recep- tors for ligands such as prolactin (PRL), growth hormone (GH), erythropoietin (EPO), and thrombopoietin (TPO) as well as to the multichain IL-3 receptor family (e.g., IL-3R and GM-CSF-R), and members of the gp130 recep- tor family. Although more than one Jak family member was suggested to interact with certain cytokine recep- tors in vitro, knockout studies in mice revealed a remark- able physiological specificity of individual Janus kinases and their associated Stat proteins (Ihle, 2001; Kisseleva et al., 2002; Levy et al., 2002). Among gene deletion models of Janus kinase family members, Jak2-deficient mice exhibit the most severe phenotype. Embryos with a complete null mutation of Jak2 die around day 12.5 of gestation due to impaired definitive erythropoiesis (Neubauer et al., 1998; Parga- nas et al., 1998). Conventional knockout models for Jak2 exhibit similarities to EPO and EPO-R-deficient mice (Wu et al., 1995), suggesting that Jak2 is essential for trans- ducing signals through the EPO receptor. In vitro differ- A. Krempler and Y. Qi contributed equally to this work. Present address for A. Krempler: Department of Medical Biochemistry and Molecular Biology, University of the Saarland, Building 44, Homburg, 66424, Germany. * Correspondence to: Dr. Kay-Uwe Wagner, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Rm. 8009, Omaha, NE 68198-6805. E-mail: kuwagner@unmc.edu Contract grant sponsor: Public Health Service, Contract grant number: CA93797 (to K.U.W.), Contract grant sponsor: National Cancer Institute, Contract grant number: CA101841 (to H.R., K.U.W.), Contract grant spon- sor: Public Health Service, National Institutes of Health, Contract grant number: DK052013 (to H.R.), Contract grant sponsor: Deutsche For- schungsgemeinschaft, Contract grant number: DFG, KR 2107/1-1 (to A.K.), Contract grant sponsor: Nebraska Cancer and Smoking Disease Research Program, Contract grant number: NE DHHS LB595 (to K.U.W.), Contract grant sponsor: Cattlemen’s Ball of Nebraska (for generation of the Jak2- deficient animal model). Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/gene.20063 © 2004 Wiley-Liss, Inc. genesis 40:52–57 (2004)