Francis Stewart

The Knockout Mouse Project

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Synthetic CpG islands reveal DNA sequence determinants of chromatin structure

The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display ‘active’ histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs. DOI: http://dx.doi.org/10.7554/eLife.03397.001

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering.

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce “unnatural” derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear‐plus‐linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05‐MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.

Structural modeling of histone methyltransferase complex Set1C from Saccharomyces cerevisiae using constraint-based docking.

Set1C is a histone methyltransferase playing an important role in yeast gene regulation. Modeling the structure of this eight‐subunit protein complex is an important open problem to further elucidate its functional mechanism. Recently, there has been progress in modeling of larger complexes using constraints to restrict the combinatorial explosion in binary docking of subunits. Here, we model the subunits of Set1C and develop a constraint‐based docking approach, which uses high‐quality protein interaction as well as functional data to guide and constrain the combinatorial assembly procedure. We obtained 22 final models. The core complex consisting of the subunits Set1, Bre2, Sdc1 and Swd2 is conformationally conserved in over half of the models, thus, giving high confidence. We characterize these high‐confidence and the lower confidence interfaces and discuss implications for the function of Set1C.

Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.

14. Engineering and Characterization of a Cloned Adenoviral-Library to Explore Natural Virus Diversity

Large double-stranded DNA (dsDNA) viruses such as adenovirus (Ad), herpesvirus, poxvirus and their recombinant counterparts were well explored in basic virology and biomedical research. Especially for the development of novel vaccines and gene therapies, Ad gained special attention and represents the most widely explored vector worldwide. Although ~70 types of human Ad and numerous nonhuman Ad (>200) have been identified so far a system for efficient Ad genome cloning and manipulation was lacking and therefore the majority of recombinant adenoviral vectors (AdVs) are based on a small fraction of Ad types. Here we report the generation and characterization of an engineered human adenoviral-library allowing exploration and system studies of the natural Ad diversity. Towards that end we first established that adenoviral genomes can be cloned and tagged in a high-throughput manner utilizing advanced homologous recombination techniques. Wild type Ads from clinical isolates including around half of the currently known adenovirus types that represent all seven human adenovirus species were propagated and direct high-throughput cloning (HTC) applied. The integrity of cloned Ad genomes was confirmed by DNA restriction enzyme pattern and virus reconstitution was conducted using optimized conditions. Most importantly next-generation sequencing (NGS) and phylogenetic analysis was performed. As a further step, half of all cloned adenoviruses representing each species were tagged with a 2A peptide-mediated multicistronic expression cassette providing a Turbo Green fluorescent protein as in vitro marker, a NanoLuc luciferase for in vivo studies and kanamycin/neomycin as selection marker. For HTC of tagged viruses the reporter cassette was inserted into the adenovirus E3 region using different orientations because the orientation of the transgene was essential for reconstitution efficiencies. After successful reconstitution these double reporters-labeled AdVs were evaluated in cultured cell lines and mouse models. In vitro characterization revealed distinct tropisms for tested viruses. Among the currently evaluated cell lines, specie B viruses demonstrated high transduction efficiencies in epithelial (Hela- and A549 cells) and endothelial cells, while Ad5 still displayed highest transduction rates in other human and murine cell types (hepatocytes, lymphocytes, neuroblastoma cells and myoblasts). For the further characterization of unknown receptor usage, we injected recombinant viruses into DSG2 or CD46 transgenic mice for in vivo bio-distribution analyses on genome level by quantitative PCR and on protein level by immunohistology analyses. We anticipate that our engineered adenoviral-library will provide a spacious novel view to the adenovirus field. As a broader perspective it will bring AdV and also other large dsDNA viruses from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology.

17. Human Adenovirus D17 Has Tropism for Endothelium Cells and Can Use Both hCAR and CD46 as Receptors

Human adenovirus type 5-derived vectors (HAdV5) from species C were broadly explored for gene therapeutic approaches and vaccination. However, disadvantages associated with this vector type are the strong liver tropism in vivo in mice, preexisting immunity and induction of robust immune responses. There is growing interest in exploring other adenovirus types of which >69 were identified. Here we constructed a new first generation adenovirus labelled with green fluorescent protein marker based on human adenovirus D17 which was first isolated from conjunctival scrapings in 1955 and aimed at characterizing the vector in vitro and in vivo. We applied a new homologous recombineering technology to construct GFP labelled early E1 gene deleted HAdV17 and HAdV5, rescued viruses in complementary stable cell lines, and then screened a panel of different cell lines by FACS analyses and quantitative PCR. Competition assays based on soluble recombinant fiber knob blocking reagents (5knob, 17knob, JO4, Augmab) were used to characterize the receptor interaction in vitro. In vivo biodistribution analyses were performed after intravenous injection of recombinant viruses into normal and CD46 transgenic mice. We observed that HAdV17 has tropism for endothelium cells which are normally refractory to HAdV5 infection. This finding was further verified using primary human umbilical vein endothelial cells (HUVEC). Moreover, after performing competition assays we found that HAdV17 can utilize both CD46 (a membrane cofactor protein which is expressed on all nucleated cells) and CAR (coxsackievirus and adenovirus receptor) as cell attachment receptors. The endothelium tropism was CD46-dependent and could be blocked by the CD46 blocking reagent Ad35K++/Augmab. In vivo biodistribution studies showed significantly increased vector genome copies (VCN) in various organs of human CD46 transgenic mice compared to normal mice indicating involvement of CD46 as a receptor. Immunohistological analyses using cell-specific marker are ongoing. Neutralizing antibody assays revealed that there was less seroprevalence with HAdV17 compared to HAdV5. In total, we believe that HAdV17-based vectors, which can use both hCAR and CD46 as receptors, hold great promise for gene therapy in endothelial disease. The understanding of the molecular interaction between virus and host will be beneficial for vaccination and drug development.

48. High-Throughput Cloning and Tagging of an Engineered Adenovirus-Library Empowers Vector Diversity for Broad Applications

Adenoviral vectors are the most widely used vectors in gene therapy, largely owing to their high efficacy in gene delivery and the ability to transduce a broad variety of cell types. Although more than 60 human adenoviruses types have been identified, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types (Ad2 and 5 from species C) and their genetic modification. Predefined tissue tropism and preexisting immunity considerably limit the application of currently used vectors, making the developing of alternative AdV s mandatory. However, generation of AdVs other than species C was hampered by the lack of convenient cloning methods, for traditional approaches were limited by the low efficiency and complicate procedure.We believe that the engineered adenovirus-library is going to provide spacious novel view to the adenovirus field. As a toolkit, it will bring the AdVs from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology studies.Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here we established a seamless recombineering pipeline for high-throughput cloning and tagging of adenoviral genomes. We amplified and purified wild type adenoviruses that represent all seven human adenovirus species. Using the recombineering pipeline we generated an engineered adenovirus-library including around half of the currently available adenovirus types. To prove integrity of the cloned adenovirus genomes we performed rescue experiments in respective cell lines permissive for adenovirus infection and we optimized rescue condition by testing different molecular forms of the adenoviral DNA (linear, circular, precise release of the DNA molecule). A limited number of selected types from each species will be tagged with a P2A peptide-mediated bicistronic-expression cassette providing a Turbo Green fluorescent protein as in vitro marker and a NanoLuc luciferase for in vivo studies. These tagged adenoviral vectors will be evaluated in cultured cell lines and mouse model. A summary of the human adenoviral vector-library which can be used for further genetic modification and tagging based on the recombineering technology is shown in figure 1.We believe that the engineered adenovirus-library is going to provide spacious novel view to the adenovirus field. As a toolkit, it will bring the AdVs from mono- to multitypes and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology studies. View Large Image | Download PowerPoint Slide