GENETICS OF SOMATIC CELLS 463 



laboratories, including our own, are continuing to characterize variations in resistance to 

 antimetabolites, with the hope that some of these may become useful genetic markers. 



The main emphasis in our laboratory is an exploration of the isoantigens of cells in 

 culture. The H-2 antigens of the mouse seem particularly promising from a geneticist's 

 point of view. These antigens are controlled by a complex locus on the ninth chromo- 

 some of the mouse and have been the subject of extensive and detailed immunogenetic 

 analysis in several laboratories for a number of years. 455, 1238 At least 20 alleles have 

 been described at this locus, and many antigenic components have been associated 

 with most of these. Thus if the H-2 antigens can be detected on cultured cells with 

 reasonable facility, a large number of markers become immediately available for 

 genetic studies in vitro by simply preparing cultures from different strains of mice. 

 Moreover, one can have confidence that the phenotypes (serotypes) of these cells bear 

 a direct relationship with the genotype. One considerable advantage a priori is the 

 possibility of using cultures derived from IR lines of Snell, or F x hybrids of these. 

 Then one can have cultures which are genetically identical except for one gene, or at 

 most a short region of one chromosome. 



Several methods of scoring the H-2 antigens of cultured cells suggest themselves 

 as possibilities. These all involve the use of isoantisera and are potentially as specific 

 as the sera which can be obtained with all the skills of the immunogeneticist. We are 

 now adapting the cytotoxic method to cultured cells. 



Cells of a DBA/2 lymphoma P-388, whose nutritional needs in vitro have recently 

 been determined, 552 are lysed when incubated with an anti-i/-2rf antiserum and 

 guinea-pig complement. This lysis has been conveniently followed with an electronic 

 cell counter. The viability of unlysed cells then can unambiguously be determined by 

 plating the culture in growth medium and counting the number of clones which 

 develop. With these procedures, we have found that these cells in long-term cultures 

 retain the H-2d phenotype of the strain from which they were derived. Continued 

 cycles of cellular killing and regrowth have selected no stable variants which have lost 

 the H-2d antigens. No tests have been performed for individual antigenic components. 



Now that the basic selection procedure has been tried, future work will be directed 

 toward attempts to select variants from an F x hybrid-derived line of cells, where loss of 

 only one dose rather than two doses are needed and to selection of variants for some 

 of the individual antigenic components. 



Means which do not result in the death of cells exhibiting an H-2 phenotype must 

 be explored. We have demonstrated that, in the absence of complement, the iso- 

 antisera are completely without effect on cellular viability. Thus labeled antibodies, 

 either fluorescent or radioactive, should be tried to score cells. The technique of 

 mixed agglutination, where red cells which share an antigen with a cultured cell are 

 coupled to such cells with antiserum, is another potentially useful method. 



To conclude, the facility with which cells can be cloned, and the complete control 

 of environment obtainable with methods of cellular culture is broadening the scope 

 and increasing the analytic power of mammalian somatic-cell genetics. 



