Introducing the constitutively active mutant of EpoR into the endogenous EpoR locus.

(A) Targeting of EpoR^R129C mutation into the endogenous EpoR locus was performed using a double replacement. In the first step, pEpoR-M2 construct was used to replace the WT allele of the EpoR to generate EpoR^hytk/− ES cells. For the second step, pEpoR^R129C construct was electroporated into the EpoR^hytk/− ES to replace the EpoR^hytk allele. Predicted restriction fragments by EcoRV digestion were indicated. (B) Southern blot analysis for knock-in event. Genomic DNA was isolated and digested with EcoRV. Southern blot was probed with an external probe A.