Use of fluorescence in situ hybridization to detect and monitor transfected and amplified sequences in recombinant CHO cells
Pallavicini, M.G.; DeTeresa, P.S.; Wurm, F.M.
Developments in Biological Standardization 70: 165-172
1989
ISSN/ISBN: 0301-5149 PMID: 2668071 Document Number: 343718
Non-radioactive fluorescence in situ hybridization was used to detect transfected and amplified sequences in recombinant Chinese hamster ovary CHO cells. The cell lines used in this study were based on dihydrofolate reductase (DHFR)-deficient CHO-DUKX cells. The recombinant CHO cells contain and express multiple copies of a constitutive DHFR expression vector, as well as an inducible Drosophila HSP 70 promoter-mouse c-myc construction as verified by Southern and northern experiments. In order to localise 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 usefulness of this method as a general tool for rapid characterisation and monitoring of recombinant cell lines is discussed.