Generation of Stable Pentraxin ExpiCHO Cell Lines

Abstract

Transfection – the introduction of foreign genetic material into eukaryotic cells – is used to study gene expression and protein production. There are two types of transfection: transient and stable. Transient transfection involves the foreign DNA being delivered into the cells’ nucleus but degraded as the cells divide. Transient needs to be performed repeatedly to maintain the finite expression, which is inefficient for long-term research. Stable transfection, where the foreign DNA integrates into the cells’ genome, is expressed for months, with the gene of interest being replicated through generations. Transient transfection is completed within three weeks, while stable needs months due to the selection screening. Along with these trade-offs of stable and transient transfection, new technologies and protocols are consistently developed, making it difficult to determine the most efficient and replicable transfection method. This project will determine this by comparing the duration and protein yield of stable and transient transfection. The procedure involves preparing the plasmids for transfection; transfecting the plasmids stably with antibiotics and cloning; transfecting the plasmids transiently; and testing for protein in the cultures. The results show production of Pentraxin I in the Zeocin transient culture was a total of 1.91 mg. Production of Pentraxin I in the Geneticin and Zeocin stable cultures was scarce and requires cloning for a substantial yield. The antibiotic selection phases are complete, with Zeocin finishing in 38 days and Geneticin in 64 days. The next steps are diluting and cloning the stable cultures; testing for protein; and comparing its yield to transient transfection.