Cytogenetic and Nuclear Studies on A9 and B82 Cells Fused Together by Sendai Virus: The Early Phase

Abstract

A9 and B82 enzyme-deficient mutants of the L line of mouse cells were hybridized artificially in vitro. After exposure of the cells to the fusing agent (inactivated Sendai virus), cultures were grown in a selective medium with aminopterin, which allows the survival only of heterokaryons which contain both A and B nuclei. The chromosome make-up of the fused cells was then studied for an initial 3-month period under various conditions. Polykaryons with 2 or more nuclei were at first observed but, as the cultures progressed, only mononucleate cells (synkaryons) persisted. Most cells in mitosis showed the chromosomal set of one A and one B cell, with an average mode and chromosome make-up very similar to, but not exactly, the sum of the average s chromosome number of one A and one B nucleus (as cells). As a rule, the synkaryons contained three ‘dot-like’ chromosomes, two of which were probably derived from the A cell and one from the B cell. In the course of the 3-month study the relative frequency of the 2s cells and their average modal number varied little. However, an increasing number of 2s cells with abnormally shaped chromosomes, in particular new ring chromosomes, was noted. Some of the cells had a mode intermediate between the 2s number and the s number (hypo-2S cells). They were thought to represent new segregants, but they were scarce, and their frequency did not increase when established fused cells were removed from the selective medium and then grown for up to 3 weeks. Nuclear polymorphism was present in all cultures and certain cytological observations gave the impression that fractions of the genomes of fused cells could be rejected through nuclear fragmentation. Synkaryons resulting from cell fusion induced by Sendai virus showed a remarkably stable chromosomal constitution for the first 3 months of culture, and were endowed with striking reproductive fitness.