A method to generate microcells from human lymphoblasts for use in microcell mediated chromosome transfer

Summary A method is described to generate microcells from human lymphobalsts 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: (a) attaching the cells to Concanvalin A coated plastic slides designed for enucleation, and (b) centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 μm in diameter were fused with thymidine kinase negative mouse fibroblast (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. This 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.