Phleomycin resistance as a dominant selectable marker in CHO cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Bleomycin and phleomycin are antibiotics used for cancer treatment.
• Genetic markers are essential for selecting cells that have successfully incorporated foreign DNA.
• The Streptoalloteichus hindustanus (Sh) ble gene is known to confer resistance to these antibiotics.

Purpose of the Study:

• To evaluate the efficacy of the Tn5 and Sh ble genes as selectable markers in mammalian cells.
• To compare the transfection efficiency and antibiotic resistance conferred by different plasmids carrying these genes.
• To assess the stability of antibiotic resistance in transfected cell lines.

Main Methods:

• Cloning of Tn5 and Sh ble genes into a mammalian expression vector under the RSV-LTR promoter.
• Transfection of Chinese Hamster Ovary (CHO) cells using calcium phosphate and polybrene-DMSO methods.
• Selection of antibiotic-resistant clones and assessment of resistance levels.

Main Results:

• Plasmids pUT506 (Tn5 ble) and pUT507 (Sh ble) were constructed and used for transfection.
• Transfection with pUT507 resulted in a higher frequency of phleomycin- or bleomycin-resistant clones.
• pUT507 transfectants exhibited greater resistance to both antibiotics and stable resistance was linked to genomic integration.

Conclusions:

• The Sh ble gene provides a more effective dominant selectable marker for CHO and other animal cell lines compared to the Tn5 ble gene.
• Stable phleomycin resistance is achieved through integration of the Sh ble gene into the host cell's genome.
• The Sh ble gene represents a valuable tool for genetic manipulation in animal cell culture.