Cell Line Identification and Characterization by Dual Targeting of Phenotypic Markers

Summary AbstractMany proposed protocols for the identification and quality control of laboratory cell lines focus on a single aspect of cell biologyThusthese approaches are unable to generate a broad biochemical profile of a cell line and are consequently unable to truly monitor a cell line for driftWehoweverintend to develop a unique protocol able to dually target both mRNA and cell surface phenotypic markers of any compositionWe propose to develop a kit that canin concert with Short Tandem Repeat analysisprobe three tiers of cell biologyDNARNAand cell surface biomarkerUsing a bacteriophagevirion we can jointly deliver both multiple copies of a targeting peptide with specificity and affinity for any type of cell surface targetas well asa distinct single stranded DNAssDNAtag incorporated into the ssDNA genome of thevirionThe targeting peptide will be genetically fused to coat protein III for display upon the surface of the virionThusbound to cell surface protein lipid carbohydrate biomarker will be utilized to translate biomarker expression into a PCR based quantifiable signalAdditionallythis PCR assessment ofbiomarkertraditional and nontraditionalcan be coupled to the analysis of mRNA sequence within the same aliquot of cellsCombining these two analyses into a single PCR based system would provide simultaneous and direct evidence of cell line identity and stabilityas well asbe able to provide initial evidence of drift due to age passage numbermedia compositionand culture conditionsWe propose development of a kit able to dually target both mRNA and any type of biomarker expressionusing patented primers specific for a panel of patentedclones and relevant mRNA transcriptsThe normalized levels of these mRNAs biomarkers qPCR qRT PCR Ct values would be reported alongside experimental dataThis added information would aid in the scientific communityandapos s ability to identify overcultured cells that need to be removed from the labfix culture conditions that deviate from normalevaluate the published dataand aid in the production of reproducible resultsdisplay has been used many times to identify new cell specific targeting peptidesIt is a versatile technique able to probe for high abundance biomarkersdirectdisplay selectionas well aslow abundance biomarkersdepletiondisplay selectionHoweverit is expected that anydisplay selection against whole cells will result in the identification of some cell specific biomarkers that are not protein products of the canonical transcription translation pathwaysWethereforepropose the use of Clustered Regularly Interspaced Short Palindromic RepeatsCRISPRknockout libraries to identify thedisplay targeted biomarkerWith this technique we will be able to identify groups of functionally related genesThusCRISPR knockout library will be useful in the identification of genes for the monitoring of non canonical biomarkerslipids carbohydratesCombining CRISPR anddisplay will allow for an unprecedented level of blindedcombinatorial investigation into all forms of biomarkersproviding an advantage over the currently availableomicstechnologies Project NarrativeWe are proposing the development of a kit able to dually target both cell surface biomarkers of any compositionprotein lipid carbohydrateand mRNA phenotypic markers of a cell line for the purpose of identifying cell typeas well ascells suffering from side effects of over culturing and altered environmentsThis work will facilitate reproducible results for a broad range of researchResearchers investigating questions of basic science to pre clinical work will all benefit from the ability to standardize cell line health and identity