INSERTIONAL INACTIVATION OF THE TK LOCUS IN A HUMAN B-LYMPHOBLASTOID CELL-LINE BY A RETROVIRAL SHUTTLE VECTOR

Insertional mutagenesis represents an inherent risk in retrovirally me diated gene therapy, but it may be a useful experimental strategy for identification and isolation of novel cellular loci. In this investiga tion we have established a model system using a heterozygous thymidine kinase (tk) marker locus in a human B lymphoblastoid cell line, and a M-MuLV based shuttle vector. The frequency of TK- mutants in cells ca rrying 1-2 proviruses per genome is approximately 2 x 10(-5), a 5-fold increase as compared to an uninfected control population. Southern an alysis of a set of 13 retrovirus infected TK- mutants revealed a predo minance of rearrangements among those mutants which had not undergone loss of heterozygosity. No consistent relationship was found to exist between the occurrence of a rearrangement and tk gene expression as de tected by northern analysis. The mechanisms of retroviral shuttle vect or insertional mutagenesis were characterized in more detail by focusi ng on a single TK- mutant, T2. The single proviral insert in T2 was fo und to lie within tk intron 2, in parallel orientation to the directio n of tk transcription. DNA sequence analysis of tk cDNA revealed the p resence of an aberrantly spliced product from which exon 4 is excluded . Aberrant splicing could sufficiently account for the low level of fu nctional tk transcript and thus the TK- phenotype in T2, although pote ntial contributions from other mechanisms cannot be excluded.