Genomic DNA-mediated gene transfer for argininosuccinate synthetase

Canavanine-resistant (Can^r) human cells overproduce argininosuccinate synthetase without the occurrence of gene amplification. Using calcium phosphate precipitation, genomic DNA from Can^r human cells was used to carry out gene transfer into Chinese hamster cells, which do not express argininosuccinate synthetase activity. Growth in tissue culture medium with citrulline substituted for arginine was adequate to select enzyme-positive colonies. Six independent isolates were selected for detailed analysis by enzyme assay, Southern blotting, Northern blotting, and S1 nuclease analysis, the last of which distinguishes human and hamster mRNA. Five isolates were transferrants containing the human structural gene and synthesizing human enzyme. One isolate represented a cell line synthesizing Chinese hamster enzyme. The data document (1) gene transfer of DNA fragments at least 80 kb in length, (2) the low level of spontaneous activation of the argininosuccnate synthetase locus in Chinese hamster cells, (3) the feasibility of this expression and selection system for DNA-mediated gene transfer, and (4) a method for distinguishing the human and hamster gene products at an RNA level.