| Popis: |
Non-radioactive fluorescence in situ hybridization was used to detect transfected and amplified sequences in recombinant CHO cells. The cell lines used in this study are based on DHFR-deficient CHO-DUKX cells. The recombinant CHO cells contain and express, as verified by Southern and Northern experiments, multiple copies of a constitutive DHFR expression vector, as well as an inducible Drosophila HSP 70 promoter-mouse c-myc construction. In order to localize and monitor the chromosomal location of transfected and amplified DHFR and c-myc sequences, biotinylated DNA probes were hybridized to metaphase preparations of several cell lines. The resulting hybrids were detected using fluorescein-linked avidin. The fluorescence signal was amplified using a biotinylated anti-avidin antibody. The number of c-myc and DHFR integration sites per metaphase, their distribution in cell populations growing at various methotrexate levels, the sizes of the amplified sequences, as well as the number of chromosomal rearrangements were measured. The results of this study will be presented and the usefulness of this method as a general tool for rapid characterization and monitoring of recombinant cell lines will be discussed. |
