QIAGEN Plasmid Plus Midi Kit (25)

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To generate targeting vectors for the human HPRT gene, 3.8 and 2.8-kb HPRT genomic fragments were PCR amplified with attB containing primers (HPRT-LH 5′Fw and HPRT 5′Rv for the 3.8-kb 5′ arm and HPRT 3′Fw and HPRT 3′Rv for the 2.8-kb 3′ arm). By using the MultiSite Gateway system (Life Technologies, Rockville, MD, USA), a floxed Hyg R or Puro R gene was placed between the 5′ and 3′ arms, thus yielding targeting vectors pHPRT-LH IRES2-Hyg and pHPRT-LH 2A-EGFP-2A-Puro. Similarly, another HPRT targeting vector, pHPRT-SH 2A-EGFP-2A-Puro, was constructed using the MultiSite Gateway system to assemble two homology arms (1.7 and 2.8-kb) and a drug-resistance gene cassette. Genomic fragments for homology arms were PCR amplified with attB containing primers (HPRT-SH 5′Fw and HPRT 5′Rv for the 5′ arm and HPRT 3′Fw and HPRT 3′Rv for the 3′ arm). All the plasmid vectors were purified with Qiagen Plasmid Plus Midi Kits (Qiagen K.K., Tokyo, Japan) and linearized with I-SceI (New England Biolabs, Ipswich, MA, USA) prior to transfection [20].

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