Proper integration of DHFR-TS knock out construct. This figure illustrates the PCR approach to determine that the knock out/-in construct has integrated into the DHFR-TS gene locus. Three different cells were tested: KI is a wildtype control, KII are cells transfected with a plasmid carrying only the disrupting cassettes but no DHFR-TS gene sequences and pKOI DVL are cells transformed with the pKOI DVL plasmid. PCR1 is a control reaction amplyfing 369 bp of the beta-hexosaminidase gene. PCR2 is to detect endogenous DHFR-TS (note that in the pKOIDVL cells there still is a wildtype gene in the MIC). PCR3 only yields PCR-product for correctly integrated pKOI DVL DNA. PCR4 shows that the full-length expression cassette has integrated. The first scheme illustrate the overall structure of the DHFR-TS gene, exons are coloured in green and introns in blue. The parts of the non-coding 5' and 3' regions of the DHFR-TS structure gene that we used as integration sites in the pKOI/pKOI DVL plasmids are coloured in grey. The next scheme gives on overall structure of the pKOIDVL expression construct that was used for transformation and integration into the MAC. The third scheme illustrates the disruption of the endogenous DHFR-TS structure gene by proper integration of the resistance and expression cassettes into the MAC.