EXPRESSION OF HUMAN AND MOUSE HPRT MINIGENES: 31

Abstract

A hypoxanthine phosphoribosyltransferase (HPRT) minigene has been constructed from both human and mouse HPRT cDNA by the addition of proper transcription initiation and polyadenylation signals from genomic subclones of the HPRT gene. Calcium phosphate mediated gene transfer of these HPRT minigenes into HPRT deletion lines has shown stable transformation frequencies from 10−7 to 5×10−7. When an enhancer from the mouse HPRT is inserted into either the mouse HPRT minigene or human HPRT minigene, the stable transformation frequency approaches 10−5. Microinjection of 5, 50, 250 linear or supercoil copies of each minigene/cell demonstrates enhancer as well as plasmid conformational importance for stable HPRT+ colonies. Enzymatic, Northern, and Southern analyses on these lines have been performed. Expression of HPRT depends on both location and copy number of the transgene. These minigenes have been inserted into retroviral vectors and shown to stably express the HPRT gene by transfection as well as infection. Titers of these helper free virus vary from 102 to 105 cfu/ml. HPRT expression in virally infected cells have been monitored by enzymatic, Northern and Southern analysis. This work was supported by a NIH Fellowship to SMC, an Arthritis Foundation Fellowship to PIP, and NIH AM31428 and the Howard Hughes Medical Institute (ACC and CTC).