A method to generate microcells from human lymphoblasts for use in microcell mediated chromosome transfer.
A method is described to generate microcells from human lymphoblasts for use in microcell-mediated chromosome transfer (MMCT). Micronuclei were induced in cells from a human lymphoblastic cell line by prolonged colcemid treatment, and were separated from these lymphoblasts by: attaching the cells to Concanavalin A coated plastic slides designed for enucleation, and centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 microns in diameter were fused with thymidine kinase negative mouse fibroblasts (LMTK-). HAT medium (hypoxanthine, aminopterin, and thymidine) was used to select microcell hybrids expressing thymidine kinase activity. Positive clones were isolated and Q-banded for chromosome analysis. Unlike previous methods, this procedure permits microcells to be easily generated from lymphoid cells. The methodology of enucleation of microcells may be extended to a variety of other donor cell types which can be micronucleated but which do not adhere tightly to enucleation slides and do not exhibit extrusion subdivision. This feature makes our methodology particularly useful for constructing a library of hybrid clones containing one or a few human chromosomes.
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods; D002877:Chromosomes, Human; D003703:Demecolcine / Q000494:pharmacology; D005821:Genetic Techniques; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007739:L Cells; D008214:Lymphocytes / Q000187:drug effects / Q000648:ultrastructure*; D051379:Mice
D007041:Hypoxanthine Phosphoribosyltransferase; D003703:Demecolcine