FIG. 4.
Primer design for allele replacement construction and deletion confirmation. Intergenic chromosomal DNA is shown as solid, and FRT DNA is hatched. (A) Chromosomal DNA with genes A, B, and C. Primers No plus Ni and Co plus Ci were used to make arms for B deletion, using chromosomal DNA as a template. R1, R2, and R3 are restriction sites. (B) The arms made in panel A are annealed at R2, reamplified, and cloned. (C) An FRT cassette is inserted at R2, and the resultant replacement plasmid is introduced into the chromosome and resolved to make a replacement. Primers Next plus F1 and Cext plus F2 are used to demonstrate that the replacement is correctly structured. (D) The chromosomal replacement after Flp-mediated cassette deletion. Arrows indicate primer position and direction.