Cytological analysis and sorting of a human supernumerary minichromosome

Abstract

In a newborn female, an abnormal karyotype, 46,XX/47,XX,+mar/47,XX,+9, was found associated with several malformations. The marker chromosome was present in 70% of peripheral blood lymphocytes, and its size appeared to be less than half of the smallest chromosomes. Several differential staining methods provided no indication as to its origin.Chromosomes isolated from EBV-immortalized lymphocytes of the patient were fractionated on a FACS-440. The marker was resolved as a sharp peak in the region close to the chromosomal debris: its DNA content seemed to be close to 40% of chromosomes 21 and 22.About 580000 minichromosomes were sorted. In order to optimize cloning conditions, a pilot cloning experiment was performed on a pool of sorted chromosomes 9, 10, 11 and 12.

Flow analysis and sorting of microchromosomes (<3 Mb)

“…The sorting of microchromosomes has been reported previously only for microchromosomes larger than 20 Mb in size (5)(6)(7)(8)20). The potential of large scale purification of MACs for gene-therapy applications (8,13,14,(20)(21)(22)(23) has generated the need for a better method for the preparation and isolation of chromosomes of smaller microchromosomes with improved purity.…”

“…Previously, flow cytometry has been applied successfully to the isolation of microchromosomes greater than 20 Mb in size (5)(6)(7)(8). To date, we are not aware of any reports on the use of flow cytometry to isolate small (<3 Mb in size), experimentally generated, microchromosomes, or mammalian artificial chromosomes, MACs (9)(10)(11)(12), which are a useful research tool for the functional characterization of genes as well as potential gene carriers for somatic gene therapy (12)(13)(14).…”

“…q 2007 International Society for Analytical Cytology Key terms: microchromosomes; mammalian artificial chromosomes; metaphase; bivariate; univariate, flow karyotype; resolution; sorting; FISH; DOP-PCR Chromosomes smaller than 20 Mb have been reported previously to occur naturally as chromosome aberrations, (1,2) and in animal species such as birds (3) and in certain species of turtles (4). Previously, flow cytometry has been applied successfully to the isolation of microchromosomes greater than 20 Mb in size (5)(6)(7)(8). To date, we are not aware of any reports on the use of flow cytometry to isolate small (<3 Mb in size), experimentally generated, microchromosomes, or mammalian artificial chromosomes, MACs (9)(10)(11)(12), which are a useful research tool for the functional characterization of genes as well as potential gene carriers for somatic gene therapy (12)(13)(14).…”