Application of microRNA Targeted 3UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools

摘要

The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3UTR targets of the let-7f miR are then cloned in the DHFR host 3UTR to determine the impact on gene expression (HMGA2 3UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3UTR and its ability to bind let-7f. The same 3UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.