Expression of the Sloppymerase™ in NIH/3T3 Cells: Exploring the Versatility of an Error Prone Fusion Polymerase

Abstract: The aim of this project is to assess the versatility of the Sloppymerase by performing stable transfection in NIH/3T3 cells and decide whether this cell line can be a candidate for further research on the Sloppymerase as a cancer model. The American cancer society predicts 1,806,590 new cancer cases in the US in 2020. To understand and by extension prevent the disease progress of cancer, proper cancer modeling is essential. The Sloppymerase is a 2-subunit fusion polymerase that has been designed by Ola Söderberg group. The Sloppymerase gene is carried by an inducible expression vector and consists of the 5’ to 3’ exonuclease subunit from Polymerase 1 and the translesion synthesis polymerase (TLS) Polymerase η. The Sloppymerase has a low fidelity and can insert mismatching nucleotides which can lead to mutations that eventually can lead to cancer. NIH/3T3 cells were grown on cell culture plates and an antibiotic kill curve was established to determine the optimal concentration of the selection antibiotics. Transfected cells were selected with Geneticin to create a stable cell line and the gene expression of the Sloppymerase was induced with Doxycycline. The RNA was extracted from cell lysate from the induced cells and was thereafter purified followed by RT-q-PCR. The protein expression was examined with Western blotting. Transcription of the Sloppymerase gene was confirmed with RT-q-PCR and stable transfection was thereby verified. No bands were seen in the Western blot and therefore protein expression has not yet been validated. Further studies are needed to examine the theoretical cancerous effects of the Sloppymerase.