Fig. 2.

Schematic plot of the pNeph-FLAG-hHO1 plasmid construction strategy. Using a 2-step sequential PCR reaction, the tag sequence FLAG was linked in frame to the 5′-end of the hHO-1 coding sequence (A). The peptide sequence of FLAG and its 5′-joining region are shown in B. FLAG-hHO-1 was inserted to the 5′-end of the murine nephrin promoter (C). The polyadenylation and intron sequences of the human β-globin gene were obtained by PCR from pTRE2 and inserted at the 3′-end of the hHO-1-coding region (C). The final pNeph-FLAG-hHO1 plasmid construct is shown in D. A 6.5-kb MluI-EcoRV fragment (E) was released from the pNeph-FLAG-hHO1 plasmid for microinjection. P1-P5 are sequences of primers used in the PCR reactions shown. Underscoring indicates key restriction sites.