Fig. 1. This diagram shows the strategy used to target the titin gene. An XhoI-SalI fragment containing the Pgk-Neo gene was cloned into the SalI site (S), as shown (black box shows position of pgk promoter). The ThK gene was cloned outside the genomic DNA as shown. (A) Diagram of the titin 10 kb genomic DNA cloned into PCRIIM. Shaded regions are the approximate positions of exons; unshaded regions are the approximate positions of introns; lines indicate bacterial vector backbone; stippled regions represent regions of the titin gene that are outside the region of genomic DNA used to generate the targeting vector; filled boxes represent the position of the promoters for the Pgk-Neo and ThK genes; the box with square hatching represents the Neo gene; the box with the dotted diamond pattern represents the ThK gene. Letters represent restriction sites as follows: E, EcoRI; H, HindIII; S, SalI; N, NotI; X, XhoI. (B) The targeting DNA was digested using a unique NotI site to produce the linearised targeting DNA as shown. This linearised DNA was electroporated into cells, where it recombined with the endogenous DNA at the genomic locus as shown, replacing the endogenous titin allele. The positions of the pair of PCR primers used for initial screening to identify targeted cells is shown, together with the position of a 1.5 kb BamHI-SalI DNA fragment used in subsequent southern analysis of the genomic DNA.