Double-minute chromosomes as megabase cloning vehicles.

Area of Science:

• Genetics
• Molecular Biology
• Mammalian Cell Culture

Background:

• Radiation-reduced chromosomes are useful for gene cloning and mapping but can lead to unwanted chromosome fusions.
• Existing methods require multiple X-ray deletion mutagenesis rounds, risking chromosomal rearrangements.

Purpose of the Study:

• To develop a novel cloning system for maintaining large mammalian DNA fragments.
• To clone Chinese hamster ovary (CHO) chromosomal DNA segments in mouse cells using double-minute chromosomes.

Main Methods:

• Developed a system using randomly inserted selectable markers for donor fragment maintenance.
• Utilized radiation-fusion-hybridization (RFH) to transfer CHO DNA fragments into mouse EMT-6 cells.
• Employed selection for G418 and methotrexate (MTX) resistance, followed by Southern analysis and in situ hybridization.

Main Results:

• Successfully cloned unrearranged Chinese hamster ovary (CHO) DNA segments of at least 600 kb.
• Confirmed the presence of cloned DNA fragments on double-minute chromosomes in mouse hybrid cell lines.
• Demonstrated the stability and maintenance of large DNA fragments without undesirable fusions.

Conclusions:

• The developed cloning system effectively maintains large subchromosomal fragments of mammalian DNA.
• This method provides a valuable tool for gene cloning and mapping, circumventing issues associated with X-ray induced chromosome chimeras.
• The use of double-minute chromosomes offers a stable platform for large DNA fragment maintenance in cultured cells.