Summary/Abstract: 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 (Ø) 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 Ø virion.
The targeting peptide will be genetically fused to coat protein III for display upon the surface of the virion. Thus,
Ø 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 Ø/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 Ø 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.
Ø 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 Ø display selection), as well as, low abundance
biomarkers (depletion Ø display selection). However, it is expected that any Ø 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 Ø 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 Ø 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.