We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe {i.e., TaqMan Probe}. This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude}. Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

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To understand a protein's function inside the cell, studies are often done to remove it using CRISPR-mediated knockout or RNAi knockdown methods. These approaches have challenges in either obtaining efficient or complete loss, are not possible if the protein target is essential for cell growth, and are genetic methods, not directly protein loss. To overcome these hurdles, we have employed a highly precise and temporally controlled target protein degradation strategy utilizing HaloPROTAC, a HaloTag Proteolysis-targeting chimera small molecule which will specifically degrade HaloTag fusion proteins in live cells. HaloPROTAC3, developed by Prof. Craig Crews at Yale University, recruits HaloTag fusion proteins to VHL E3 ligase complexes, resulting in ubiquitination and degradation of the HaloTag fusion via the ubiquitin-proteasomal pathway. For phenotypic studies, removal of the endogenous proteins is often required, therefore we have developed efficient protocols for the introduction of HaloTag via CRISPR/Cas9 gene editing into either the endogenous N- or C-terminal loci of any target protein. We have additionally appended the 11 amino acid HiBiT to the HaloTag-target protein, allowing for highly quantitative and kinetic monitoring of degradation to be tracked in live cells without the use of antibodies. Shown here are several key therapeutic proteins, BRD4 and B-catenin, which have been endogenously tagged with HiBiT-HaloTag via CRISPR/Cas9 and show rapid and sustained degradation (80-90% loss) after treatment with HaloPROTAC3. For BRD4, this loss was similar to what was observed using a BET-family specific PROTAC. For B-catenin, further phenotypic studies were done to show a muted response to Wnt3a stimulation and TCF/LEF promoter gene activation after degradation of endogenous B-catenin. These examples demonstrate that the technologies can be used readily to elicit robust degradation of target proteins, with control over the protein level and the time frame for which the protein will be lost, in order to more fully understand protein function. Together, these provide new opportunities for phenotypic studies based directly on control of protein levels and loss, with the ability to study the roles of essential proteins not amenable to genetic knockout or knockdown approaches. Citation Format: Elizabeth A. Caine, Sarah Mahan, Kristin Riching, Nancy Murphy, Mark McDougall, Cesear Corona, Chris Heid, Danette L. Daniels, Marjeta Urh. Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag technologies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6409.