Figure 6

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 D_VL are cells transformed with the pKOI D_VL 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 pKOID_VL cells there still is a wildtype gene in the MIC). PCR3 only yields PCR-product for correctly integrated pKOI D_VL 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 D_VL plasmids are coloured in grey. The next scheme gives on overall structure of the pKOID_VL 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.