Generation of a novel Jak2-YY1007/1008FF activation-loop mutant mouse model. (A) The murine Jak2 gene is depicted schematically at the top. The Ω-replacement vector was designed to replace exon 21 of the wild-type gene with a mutated version (YY1007/1008/FF). For positive selection, a neo cassette was used, whereas an HSV-Tk cassette was used for negative selection. The 5′ short arm was cloned using a XbaI restriction site and the 3′ short arm was inserted via BamHI. The targeting vector was electroporated into E14.1 ES cells.⁵  The neomycin resistance cassette was deleted by crossing Jak2-FF mice with a deleter mouse strain (N = NcoI). (B) For Southern blot analysis, DNA was digested with NcoI. Wild-type and Jak2-FF–targeted ES cells (lanes 1 and 4) and embryos after germline transmission of the Jak2 mutation (lane 2) and wild-type (lane 3) were analyzed. The expected fragment size after hybridization with the flanking probe is approximately 7.7 kb for the wild-type and approximately 5.5 kb for the targeted allele. Lane 1, Jak2^+/+ ES cell; lane 2, Jak2^+/+ mouse; lane 3, targeted Jak2^+/FF mouse; lane 4, targeted Jak2^+/FF ES cell clone 92/46. (C) Screening PCR of targeted ES cell clones and mice after deletion of the neo cassette via cross with a cre-deleter mouse line. Genomic DNA of ES cells and mice was used in a PCR with primers 1 und 2 (see Methods). Lane 1, Jak2^+/+ control; lane 2, targeted Jak2^+/FF mouse; lane 3, targeted Jak2^FF/FF mouse; lane 4, Jak2^+/+ control ES; lane 5, Jak2^+/FF ES cell clone 92/46; lane 6, H₂O control without template DNA. (D) Western blot analyses of protein extracts from MEFs generated from E12.5 with the genotype Jak2^+/+, Jak2^FF/FF, and Jak2^−/−. Blots were incubated with antibodies directed against Jak2, Stat1, and β-actin.