Our lab is interested in developing new genetic technologies with applications for gene discovery and human health. Some of the technologies we are currently exploring are genetic screening with RNAI and ORF libraries, autoantibody discovery, and new methods for antibody discovery. Currently, we are particularly interested in auto-immune diseases and vaccine design. Some of our work is detailed below.
We, together with our collaborators in Greg Hannon’s laboratory, have generated large libraries of shRNAs that cover the entire human and mouse genomes (171, 183). The libraries are designed using shRNA prediction algorithms and synthesized in a massively parallel fashion using Agilent microarrays. Oligonucleotides are PCR amplified and cloned into retroviral or lentiviral vectors. At this point they can either be used directly or sequence verified and arrayed. Each vector also has a unique barcode associated with it for microarray deconvolution of pools. In this way we can generate large libraries of shRNAs for genetic screening (see the Technology Development Section for more details).
New shRNA vectors, the PRIME Vectors
The advent of RNAi has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA from polymerase III promoters can be encoded in transgenes and used to produce siRNAs that downregulate specific genes. However, we discovered that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a micro-RNA context (182). Importantly, our shRNA expression system (called PRIME vectors) allows for the multicistronic co-transcription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these PRIME vectors will facilitate genome-wide loss-of function screens and is an important step towards using bar coding strategies to follow loss of specific sequences in complex populations.
We have also developed a series of PRIME vectors that show better knockdown in mouse ES cells and other cells where the CMV promoter might not be optimal. In addition, we have developed more tet-responsive inducible vectors that encode their own reverse tet-activator.
Recombinant DNA Technologies
MAGIC: An in vivo genetic method for the rapid construction of recombinant DNA
We developed a novel, highly engineered in vivo cloning method, MAGIC (Mating Assisted Genetically Integrated Cloning), that facilitates the rapid construction of recombinant DNA molecules (180). MAGIC employs bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze DNA fragment transfer between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of novel regulatory elements with high efficiency. MAGIC eliminates the need for restriction enzymes, DNA ligases, preparation of DNA and all in vitro manipulations required for subcloning and allows the rapid construction of multiple constructs with minimal effort. We demonstrate MAGIC can generate constructs for expression in multiple organisms. As this new method requires only the simple mixing of bacterial strains, it represents a significant advance in high throughput recombinant DNA production that will save researchers significant amounts of time, effort and expense in functional genomics studies.
SLIC Cloning: a method for subcloning without restriction enzymes or DNA ligase
We developed a novel cloning method SLIC (Sequence and Ligation-Independent Cloning) that allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing (195). SLIC mimics in vivo homologous recombination by relying on exonuclease generation of single strand DNA (ssDNA) overhangs on insert and vector fragments and the assembly of these fragments by recombination in vitro. SLIC inserts can be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and is much more sensitive when combined with RecA to catalyze homologous recombination. It also provides a new method for site-directed mutagenesis of a gene. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
Enhanced display technologies for biomarker discovery – PhIP Seq
The sensitive detection of circulating biomarkers represents a powerful approach to screening for early malignancy. However, current techniques are limited in their ability to distinguish normal from pathological states. This work aims to improve on current screening technology, utilizing novel principles in synthetic biology. In particular, we combine next generation DNA sequencing with the display of polypeptide libraries encoded by microarray-derived oligonucleotides. Using the T7 bacteriophage system, we are able to display the complete human peptidome and thereby seek to discover autoantigen biomarkers common to breast cancer patients. In parallel we are developing a fully defined, synthetic human single chain variable fragment (scFv) antibody library. This library will be used in ribosome display format for a scFv library-versus-peptide library screen in an effort to create a proteomic scale scFv set. Our hope is that development of these technologies will facilitate the identification of circulating cancer biomarkers, thereby enabling early diagnosis and treatment of the disease.
The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo.
The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.
Lambda YES cDNA Expression System
We have developed a multifunctional lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of E. coli and S. cerevisiae mutations (23). Lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage l to plasmid clones using the cre-lox site-specific recombination system, referred to as automatic subcloning. Lambda YES vectors utilize an adaptor selection for inserts and can generate libraries with 109 recombinants per mg of cDNA insert. cDNA libraries constructed in these vectors have been used to isolate genes from humans by complementation of yeast. Using this technology we isolated the human Cdk2 gene (24) by complementation of a yeast cdc28 Ts mutation and the Cyclin F gene (40) as a suppressor of cdc4 Ts mutations which led to the discovery of F-box proteins (77).
UPS – the Univector Plasmid Fusion System: A method for rapid construction of recombinant DNA molecules without restriction enzymes
Modern biological research is highly dependent upon recombinant DNA technology. The functional analysis of a single gene requires the introduction of that gene into multiple vectors for different purposes. Conventional cloning methods are time-consuming and individual cloning events must be performed independently. To approach solving this problem, we sought to develop a systematic and uniform method with which to manipulate large sets of genes.
We developed a series of novel cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules (102). The central method is named the Univector Plasmid-fusion System (UPS). UPS employs cre-lox site-specific recombination to catalyze plasmid fusion between the Univector, a plasmid containing the gene of interest, and host vectors containing regulatory information. Fusion events are genetically selected and result in placement of the gene under the control of novel regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the Univector into a host vector. UPS eliminates the need for restriction enzymes, DNA ligases, and many in vitro manipulations required for subcloning and allows the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can be used to transfer whole libraries into new vectors. New methods for directional cloning of PCR fragments and for the generation of epitope tags and other fusions at the 3′ end of genes using homologous recombination in E. coli are described that facilitate cloning and manipulation of genes in the Univector.
Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, an ability required for future genomic analysis. We are continuing to develop large-scale capabilities using UPS as an alternative to the expensive and limited GATEWAY system.
Advances in the Two-Hybrid System
The two-hybrid cloning system is a powerful genetic method to identify genes via protein-protein interactions. We have made a number of improvements to this method which are designed to increase the number of potential protein-protein interactions detectable and to streamline the labor intensive process of authenticating clones identified in the primary screen. The first improvement was the design of genetic selections instead of screens to detect interacting proteins (32, 39). We developed the strain Y190 using HIS3 as a reporter and Y166 which employed a URA3 reporter for this purpose. A selection vastly increases the numbers of library clones that can be searched and is now built into all systems. Secondly, through the use of negative selections for plasmid loss coupled with a replica-mating strategy, we have developed a rapid method to distinguish between the genuinely interacting clones and the nonspecific background that is inherent to the system. Third, a lambda phage vector, lambda ACT2, was made that allows construction of highly complex, HA epitope-tagged, directional libraries of GAL4 activation domain-fused cDNAs that can be converted to plasmid form by in vivo cre-lox mediated site-specific recombination. We also introduced yeast mating of library clones to baits as a rapid way to both generate libraries and well as to counter screen against false positives. These methods have been incorporated into all current two-hybrid system kits. We used these methods to identify the human p21 and p57 Cdk inhibitors.
A Cytoplasmic Two Hybrid System, The SOS Recruitment System
In collaboration with Michael Karin’s lab, we have developed a novel two hybrid system for the cloning of genes involved in protein-protein interactions (84). This system, called the SOS Recruitment System, SRS, relies on the activation of the ras signalling pathway when a fusion protein to the ras guanine nucleotide exchange factor, SOS, is recruited to the inner surface of the plasma membrane by physical association with a second protein targeted to the same membrane by a myristylation sequence. When SOS is successfully recruited to the plasma membrane, it activates ras and bypasses the need for the endogenous exchange factor, Cdc25, an essential protein. This system allows protein-protein interactions to be detected in the cytoplasm. Furthermore, proteins that activate transcription can be used in this system giving it an advantage over the transcriptionally based two-hybrid system. We have developed a lambda-based expression vector, lambda MS-TRP for expression of myristylation-fused cDNA libraries an other useful vectors for this method. Visit our protocols page for more information