Double-minute chromosomes as megabase cloning vehicles.
Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.
Hahn
P J
PJ
Department of Radiology, State University of New York Health Science Center, Syracuse 13210.
Giddings
L
L
Longo
J
J
Lane
M J
MJ
Scalzi
J
J
Hozier
J
J
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D002872:Chromosome Deletion; D002875:Chromosomes / Q000528:radiation effects / Q000648:ultrastructure; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D005784:Gene Amplification; D005822:Genetic Vectors; D006822:Hybrid Cells / Q000648:ultrastructure; D008322:Mammals / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D012641:Selection, Genetic