Linearized targeting vector was transfected into 129Sv embryonic stem (ES) cells (GenOway, Lyon, France) according to GenOway’s electroporation procedures. Polymerase chain reaction (PCR), Southern blot, and sequence analysis of G-418–resistant ES clones revealed the recombined locus in two clones. PCR across the 5′ end of the targeted locus used a forward primer hybridizing upstream of the 5′ homology arm (5′-ATAGCGTTGGCTACCCGTGATATTGC-3′) and a reverse primer hybridizing within the neomycin cassette (5′-AGGCTAGGCACAGGCTACATCCACAC-3′). Two Southern blot assays were hybridized with an internal and an external probe to assess recombination accuracy at the respective 5′ and 3′ ends of the Kcc2 locus. The absence of off-target mutations was confirmed by sequence analysis.

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