Novel Expression Methodology
Genome-editing technologies now permit precise positioning of deletions, modifications, and transgenes within living cells. These ideas have led Zsolt Keresztessy, Ph.D., senior research fellow at Proxencell, to a method employing sequence-specific meganucleases and TAL effector nucleases to generate stable monoclonal cell lines expressing membrane-bound antigens, FCGR receptors, and monoclonal antibodies. TAL effector nucleases are novel sequence-specific nucleases, formed by fusing a transcription activator-like (TAL) effector DNA binding domain to the catalytic head of an endonuclease.
As a core facility for the University of Debrecen in Hungary, Proxencell provides protein expression services using optimized synthetic genes and expression organisms that include bacteria, yeast, and both insect and mammalian cells for small- and large-scale protein production.
Dr. Keresztessy explains that specific genome editing technologies are still in the initial evolutionary phase—true especially for TAL effector nucleases. “That means, in addition to requiring substantial optimization work, investigators must also innovate in the adaptation of commercially available systems from, for example, Cellectis Bioresearch or Life Technologies.”
Uncovering effective ways to transfer and express sequence-specific nucleases (e.g., plasmid DNA, mRNA, or proteins) into your target cells or cell lines, together with accessory sequences including like templates for homologous recombination or genome editing reporter constructs, is critical.
“As a result, we were forced to develop new technologies for assessing genome modifications at early stages of TAL transfections, strategies and tools for detecting and enriching knockout cells, and new approaches for mapping TAL specificity in vivo in automated and high-throughput assays.”
Dr. Keresztessy and colleagues have enjoyed several successes in designing bioassay-worthy cells through these strategies. More relevant here are production cells. When the goal is overexpressing a protein—for example, a monoclonal antibody—for large-scale production, a variety of choices exist from commercially available reagent kits. Dr. Keresztessy’s system of choice is the cGPS CHO-Sa CEMAX system from Cellectis.
“However, we needed to construct our own version of the integration matrix vector provided by the vendor to efficiently express our mutant therapeutic antibodies, which we aimed to use as cellular assay controls in our research applications. Into the vector, we cloned the synthetic genes of the light and heavy chains of a human IgG framework, with the desired mutations, linked via an in-house designed and optimized IRES sequence.”
A total of 20 unique restriction endonuclease sites were engineered into genes to allow subcloning of variable regions for future applications.
Dr. Keresztessy sees great potential in sequence-specific nuclease-assisted genome editing for cell-line development. Applications include basic research for functional studies using reporters with gene tagging, promoter and enhancer modifications, and gene disruptions. The general technique could be employed to produce more potent reporter effector cell lines incorporating molecular sensory systems for the specific detection of macrophage activation, apoptosis, necrosis, differentiation, and others for cell-based assays.
“In my opinion one of the main limitations of the technology is efficiency variations for various cell types,” Dr. Keresztessy says. “The cost of specific genome modifications may also be limiting for academia and research institutes.”