Many proposed protocols for the identification and quality control of laboratory cell lines focus on a single aspect of cell biology. Thus, these approaches are unable to generate a broad biochemical profile of a cell line and are consequently unable to truly monitor a cell line for drift. We, however, intend to develop a unique protocol able to dually target both mRNA and cell surface phenotypic markers of any composition. We propose to develop a kit that can, in concert with Short Tandem Repeat analysis, probe three tiers of cell biology; DNA, RNA, and cell surface biomarker. Using a bacteriophage () virion we can jointly deliver both multiple copies of a targeting peptide with specificity and affinity for any type of cell surface target, as well as, a distinct single stranded DNA (ssDNA) tag incorporated into the ssDNA genome of the virion. The targeting peptide will be genetically fused to coat protein III for display upon the surface of the virion. Thus, bound to cell surface protein/lipid/carbohydrate biomarker will be utilized to translate biomarker expression into a PCR based quantifiable signal. Additionally, this PCR assessment of /biomarker (traditional and non- traditional) can be coupled to the analysis of mRNA sequence within the same aliquot of cells. Combining these two analyses into a single PCR based system would provide simultaneous and direct evidence of cell line identity and stability, as well as, be able to provide initial evidence of drift due to age/passage number, media composition, and culture conditions. We propose development of a kit able to dually target both mRNA and any type of biomarker expression, using patented primers specific for a panel of patented clones and relevant mRNA transcripts. The normalized levels of these mRNAs/biomarkers qPCR/qRT-PCR Ct values would be reported alongside experimental data. This added information would aid in the scientific community's ability to identify overcultured cells that need to be removed from the lab, fix culture conditions that deviate from normal, evaluate the published data, and aid in the production of reproducible results. display has been used many times to identify new cell specific targeting peptides. It is a versatile technique able to probe for high abundance biomarkers (direct display selection), as well as, low abundance biomarkers (depletion display selection). However, it is expected that any display selection against whole cells will result in the identification of some cell specific biomarkers that are not protein products of the canonical transcription/translation pathways. We, therefore, propose the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout libraries to identify the display targeted biomarker. With this technique we will be able to identify groups of functionally related genes. Thus, CRISPR knockout library will be useful in the identification of genes for the monitoring of non-canonical biomarkers (lipids/carbohydrates). Combining CRISPR and display will allow for an unprecedented level of blinded, combinatorial investigation into all forms of biomarkers, providing an advantage over the currently available ?-omics? technologies.
We are proposing the development of a kit able to dually target both cell surface biomarkers of any composition (protein/lipid/carbohydrate) and mRNA phenotypic markers of a cell line for the purpose of identifying cell type, as well as, cells suffering from side-effects of over-culturing and altered environments. This work will facilitate reproducible results for a broad range of research. Researchers investigating questions of basic science to pre-clinical work will all benefit from the ability to standardize cell line health and identity.
