A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50 000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation

Genomic analysis by single cell flow sorting

Thesis (Ph. D.)--University of Washington, 2003The Human Genome Project has dramatically changed the landscape of biology. With the availability of genomic sequence from humans and many other organisms, new biological questions are being asked that involve the simultaneous study of thousands of genes or proteins. The invention of new technologies continues to be important for the timely investigation of many of these questions.In this work, we present new technologies that address several genomcs-level questions using electronic cell sorters. Because these machines are capable of examining and sorting tens of thousands of cells per second, they are potentially ideal platforms for investigating large systems. The challenge lies in converting biological attributes into readable physical attributes. In this work, we present the development of a series of plasmid vectors that encode biological states as the ratio of two fluorescent proteins in E. coli.Using this doctrine, we created the pGRFP series of vectors that can be used to rapidly isolate insert-bearing clones on an electronic cell sorter. This technique is a powerful alternative to traditional colony picking based on blue/white color selection. The speed of the electronic cell sorter allows us to deposit single cells into tubes as fast as the tubes can be transported. We validate this method's precision is selecting insert-bearing clones and show its usefulness in a small sequencing project.We also show how the pGRFP series vectors can be used to classify a large number of protein mutants. We sequenced hundreds of active mutants of a human enzyme. From these data, we introduce the concept of the "x-factor" that indicates a particular protein's tolerance to mutation. We are able to make striking correlations between the pattern of mutability throughout the enzyme and what is known about its 3D structure and mechanism of action.Finally, we present the pGFPpDsRed series of vectors that show promise in detecting DNA-Protein interactions. This might make a very useful tool for scanning genomic DNA for transcription factor binding sites on the road to solving regulatory networks. Conversely, a large number of protein mutants could be searched quickly to find variants that bind to a specific DNA sequence

Human immunodeficiency virus type 1 biological variation and coreceptor use: from concept to clinical significance.

There is ample evidence for intra-patient evolution of the human immunodeficiency virus type 1 (HIV-1) biological phenotype during the pathogenic process. Evolution often involves switch of coreceptor use from CCR5 to CXCR4, but change to more flexible use of CCR5 occurs over time even in patients with maintained CCR5 use. The increasing use of entry inhibitors in the clinic, often specific for one or the other HIV-1 coreceptor or with different binding properties to CCR5, calls for virus testing in patients prior to treatment initiation. Cell lines expressing CCR5/CXCR4 chimeric receptors are tools for testing viruses for mode of CCR5 use. It is conceivable that small-molecule entry inhibitors that differentially bind to CCR5 can be matched for best effect against HIV-1 with different modes of CCR5 use, thereby allowing an individualized drug choice specifically tailored for each patient