Although gene targeting strategies are well established in single-cell eukaryotic models, the absence of a valid and efficient method for material derived from multi-cellular organisms such as birds or mammals has limited their use. However, Orlowska and colleagues (July 2013) have developed a new approach involving novel pQuant constructs in tandem with LC-MS proteomic analysis to examine multi-gene targeting in the chicken B-lymphocyte cell line DT40.1
DT40 has a high rate of homologous recombination coupled with an extremely short doubling time in culture (approximately 11 hours). It is, therefore, an excellent candidate for gene targeting work. It also grows in suspension, making it an ideal candidate for a high-volume flow cytometry analysis. However, the lack of efficient strategies for creating multiple knock-in/knock-out genes, combined with an absence of suitable reference libraries, means that this cell line’s amazing properties have not been fully tapped for proteomics research.
Using customized pQuant constructs and adapting existing proteomics analytical techniques, Orlowska and colleagues developed a method with the potential for scaling up the transfection of DT40 cells. They propose this method can be applied to multi-gene experiments.
Custom-made pQuant constructs for genes under investigation were prepared by a sequence- and ligation-independent cloning (SLIC) approach, using the Quant peptide, a TEV protease cleavage site and then protein-A, FLAG or EGFP for tagging the endogenous protein. Researchers demonstrated the functionality of the vectors and the experimental procedure by targeting four genes — EXOSC8, EXOSC9, CNOT7 and UPFI — known to play roles in RNA metabolism.
Following transfection, flow cytometry confirmed DT40 clones generated via genetic targeting. Fluorescence microscopy further confirmed the success of the procedure, confirming, as expected, that protein abundance fluctuates with cell cycle events. Furthermore, live microscopy of transfected cells showed that the gene products localized to the defined intracellular compartments relevant to protein function. Polyribosome profiling further confirmed the subcellular fractions involved.
Proteomic analysis started with co-immunoprecipitation of protein partners from cell lysates. Resulting materials were confirmed by gel electrophoresis and liquid chromatography–mass spectrometry (LC-MS) analysis using an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Data resulting from LC-MS were analyzed using Andromeda/MaxQuant software with reference to various databases, including a UniProt database containing Gallus gallus sequences. The researchers also generated a database from DT40 transcriptome mapping and deduced chicken homologs to previously studied proteins.
MS data analysis showed both protein identity and abundance, as well as protein–protein interactions. The authors conclude that their gene targeting methods did not interfere with biological function of the products under scrutiny. Although they note that results might not accurately reflect one of their constructs (EXOSC9) — citing a degradation issue interfering with MS — the authors also conclude that this novel technique is suitable for investigation of mammalian gene targeting.
1. Orlowska, K.P., et al. (2013, July 27) “A new strategy for gene targeting and functional proteomics using the DT40 cell line,” Nucleic Acids Research [epub ahead of print] (pp. 1–13), doi: 10.1093/nar/gkt650.
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