Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes

Abstract

We cotransformed mouse 3T3 cells with total genomic human DNA and the dominant selectable bacterial gene Neoand analyzed 121 Neo^R clones for expression of 15 human “housekeeping” enzymes which can be distinguished from their murine homologs. The estimated frequency of expression of unlinked human genes was 1 in 360 Neo^R clones and at least three different human enzymes (peptidase D, phosphoglucomutase 1, and acid alpha glucosidase) were detected. We further examined the frequency and stability of cotransformation for one of these enzymes, acid alpha glucosidase (GAA). We tested approximately 4000 Neo^R clones and found 25 clones expressing human GAA, as determined by rocket immuno-electrophoresis (RIE) specific for human GAA. Transformants progressively became negative on continued growth and retesting by RIE, with only two clones still expressing GAA at the eighth testing. This apparent loss of expression was not due to nonclonality of the original isolates. In one subclone examined, loss of expression was accompanied by loss of both Neo -derived pBR322 and human Alu repetitive sequence DNA. Thus, under the conditions utilized, cotransformants expressing homomeric housekeeping enzymes were found at relatively high frequency but were progressively lost even under conditions selective for expression of the dominant vector.