अमूर्त

A new technique for constitutive high-level production of heterologous proteins in eukaryotic systems using the CRISPR/Cas9 system was usedto modify the genome of a Hybridoma cell line

Jafar Mammadova

In several domains of biology, the CRISPR/Cas9 system's capability has revolutionised genome editing. In recent years, many applications, notably those using protein expression technology, have grown at an exponential rate. Because the CRISPR/Cas9 system prevents haphazard gene integration, it can be used to create a stable cell line for high-yield recombinant protein expression. We present a strategy for leveraging the CRISPR/Cas9 system to alter a hybridoma cell line for constitutive expression of proteins of interest. To begin, we substituted part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, resulting in a precise knock-in in the hybridoma genome with the goal of optimising the approach. GFP expression and secretion into the cells were confirmed. The gene encoding a protein of diagnostic relevance, the Bovine Herpesvirus 1 glycoprotein E, was then inserted in the donor DNA using the same method. We were able to isolate a viable clone that secreted gE protein fused to GFP into the culture medium. ELISA and Western Blot tests verified this result. This investigation supports the suitability of this cell line for the synthesis of diagnostically important proteins in a mammalian system through sustained gene expression. These tests will allow the technique to progress from proof-of-concept to more targeted applications in infectious illness detection