Recombination-mediated Cassette Exchange Research Articles

Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange and drug selection.

I outline a streamlined method to insert large, single-copy transgenes into the Caenorhabditis elegans genome using recombination-mediated cassette exchange (RMCE) that relies solely on drug selection yielding a homozygous fluorescent protein (FP) marked transgene in 3 generations (8 days) at high efficiency (>1 insertion per 2 injected P0 animals). Landing sites for this approach are available on four chromosomes in several configurations which yield lines marked in distinct cell types. An array of vectors permit creating transgenes using a variety of selection methods (HygR, NeoR, PuroR, and unc-119) that yield lines expressing different colored FPs (BFP, GFP, mNG, and Scarlet). Although these transgenes retain a plasmid backbone and a selection marker, the inclusion of these sequences typically does not alter the expression of several cell-specific promoters tested. However, in certain orientations, promoters exhibit crosstalk with adjacent transcription units. In cases where crosstalk is problematic, the loxP-flanked fluorescent marker, plasmid backbone, and hygR gene can be excised by crossing through germline Cre expressing lines also created using this technique. Finally, genetic and molecular reagents designed to facilitate customization of both targeting vectors and landing sites are also described. Together, the rapid RMCE toolbox provides a platform for developing further innovative uses of RMCE to create complex genetically engineered tools.

A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

Murine cancer cell lines are powerful research tools to complement studies in genetically engineered mouse models. We have established 21 melanoma cell lines from embryonic stem cell-genetically engineered mouse models driven by alleles that model the most frequent genetic alterations in human melanoma. In addition, these cell lines harbor regulatory alleles for the genomic integration of transgenes and the regulation of expression of such transgenes. In this study, we report a comprehensive characterization of these cell lines. Specifically, we validated melanocytic origin, driver allele recombination and expression, and activation of the oncogenic MAPK and protein kinase B pathways. We further tested tumor formation in syngeneic immunocompetent recipients as well as the functionality of the integrated Tet-ON system and recombination-mediated cassette exchange homing cassette. Finally, by deleting the transcription factor MAFG with an inducible CRISPR/Cas9 approach, we show the utility of the regulatory alleles for candidate gene modulation. These cell lines will be a valuable resource for studying melanoma biology and therapy.

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.

PhiC31 integrase for recombination-mediated single-copy insertion and genome manipulation in Caenorhabditis elegans.

Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

Using recombinase-mediated cassette exchange to engineer MIN6 insulin-secreting cells based on a newly identified safe harbor locus.

Aims/IntroductionRecent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin‐secreting MIN6 cells.Materials and MethodsMIN6 cells were first engineered to have a tetracycline‐regulated expression system. Then, we used the recombination‐mediated cassette exchange strategy to explore the silencing‐resistant site in the genome and generated a master cell line based on this site.ResultsWe identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt‐based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over‐ or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively.ConclusionsWe engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion.

Generation of Drosophila attP containing cell lines using CRISPR-Cas9.

The generation of Drosophila stable cell lines has become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here, we describe a toolkit and procedure that combines CRISPR/Cas9 and theaaa PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.

Additional Landing Sites for Recombination-Mediated Cassette Exchange in C. elegans.

Recombination-mediated cassette exchange (RMCE) is a recently developed alternative method for creating single copy transgenes using recombination rather than repair of double stranded breaks as the mechanism for driving integration into the genome. Two alternative methods for performing RMCE have been developed; a two-component approach using an unlinked source of FLP recombinase, and a one-component approach using a FLP expression cassette within the landing site. Here, I describe new landing sites for performing both types of RMCE. The new landing sites are located within 50 bp of well-vetted MosSCI insertion sites on Chr II and Chr IV.

Abstract A17: A versatile mouse-modeling platform for rapid in vivo melanoma studies

Abstract Melanoma accounts for about 1% of skin cancers but causes the majority of skin cancer-related deaths. Despite progress in treating melanoma with immunotherapy and targeted therapy, melanoma patients still suffer from relapse due to resistance to these treatments. A better understanding of the molecular mechanisms that contribute to melanomagenesis and resistance may provide novel avenues for the development of improved treatment strategies. To accelerate the speed, increase the throughput, and lower the cost of delineating such mechanisms in vivo, we developed a novel embryonic stem cell-genetically engineered mouse-modeling (ESC-GEMM) platform. We generated ESC lines harboring twelve combinations of four common driver mutations (BRAF-V600E, NRAS-Q61R, loss of PTEN, loss of CDKN2A) that can be efficiently targeted by recombination-mediated cassette exchange (RMCE) to introduce inducible alleles of interest. Importantly, all of our ESC lines are capable of producing high-contribution chimeras, which exhibit the same melanoma kinetics and overall survival as conventionally bred mice. Thus, our platform offers readily adaptable melanoma models whose melanoma phenotypes range from long latency/low penetrance to aggressive melanoma with complete penetrance. We employed our ESC-GEMM approach to compare the efficiency of gene depletion by conditional knockout, CRISPR/Cas9, or RNAi. We found that CRISPR/Cas9 depletion of Pten on a Braf-V600E background produces fewer melanomas but with nearly the same latency as conditional knockout using the Cre-lox system. Silencing of Pten with inducible shRNA resulted in significantly increased latency as well as in melanomas with a distinct macroscopic appearance and widespread lymph node metastases. While inducible shRNAs allowed us to restore Pten expression in established melanomas, depletion of Pten with inducible CRISPR enabled us to control the number of moles/melanomas by limiting the duration of Cas9 expression. We also used inducible CRISPR to sequentially activate Braf-V600E and delete Pten, which more closely mimics the sequence of events in human melanomas. Interestingly, while sequential introduction of these genetic changes had no effect on the overall survival, a subset of mice developed a significantly greater number of tumors. Finally, we established melanoma cell lines from untargeted chimeras, which can be targeted with Tet-inducible expression cassettes and transplanted into syngeneic hosts. Taken together, we have established a speedy mouse-modeling platform that will stimulate and accelerate in vivo melanoma research, provide a powerful resource to investigate the pathobiology of melanomagenesis, and allow the genetic and pharmacologic evaluation of novel treatments. Citation Format: Ilah Bok, Jordan Reff, Arianna Nenci, Jose G. Gonzalez, Florian A. Karreth. A versatile mouse-modeling platform for rapid in vivo melanoma studies [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A17.

Generating and working with Drosophila cell cultures: Current challenges and opportunities.

The use of Drosophila cell cultures has positively impacted both fundamental and biomedical research. The most widely used cell lines: Schneider, Kc, the CNS and imaginal disc lines continue to be the choice for many applications. Drosophila cell lines provide a homogenous source of cells suitable for biochemical experimentations, transcriptomics, functional genomics, and biomedical applications. They are amenable to RNA interference and serve as a platform for high-throughput screens to identify relevant candidate genes or drugs for any biological process. Currently, CRISPR-based functional genomics are also being developed for Drosophila cell lines. Even though many uniquely derived cell lines exist, cell genetic techniques such the transgenic UAS-GAL4-based RasV12 oncogene expression, CRISPR-Cas9 editing and recombination mediated cassette exchange are likely to drive the establishment of many more lines from specific tissues, cells, or genotypes. However, the pace of creating new lines is hindered by several factors inherent to working with Drosophila cell cultures: single cell cloning, optimal media formulations and culture conditions capable of supporting lines from novel tissue sources or genotypes. Moreover, even though many Drosophila cell lines are morphologically and transcriptionally distinct it may be necessary to implement a standard for Drosophila cell line authentication, ensuring the identity and purity of each cell line. Altogether, recent advances and a standardized authentication effort should improve the utility of Drosophila cell cultures as a relevant model for fundamental and biomedical research. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes.

Rapid Chimerization of Antibodies.

We previously developed the in vitro method to generate monoclonal antibodies (mAbs) from libraries constructed with chicken B-cell line DT40 (referred to as the "ADLib system"). As the wild-type DT40 cells express immunoglobulin M (IgM), the original ADLib system provides monoclonal antibodies in chicken IgM format. For the therapeutic, diagnostic, and research purposes, the Fc regions of IgMs should be exchanged to other classes and species, for example human or murine IgG. However, the Fc engineering by conventional bioengineering process is laborious and takes plenty of time. Here, we developed a method to enable the seamless replacement of the Fc regions of antibodies generated by the ADLib system, using recombination-mediated cassette exchange (RMCE). In this system, two Cre recombinase recognition sites were inserted into the IgM's Fc region of the DT40 genome, allowing the exchange of the Fc region to the sequences of interest by co-transfection of a donor sequence and a Cre recombinase expression vector. We describe the detailed protocol of the technology: how to construct the RMCE host strains, select mAbs by the ADLib system, and exchange their Fc regions to generate chimeric mAbs.

Abstract A08: Fast and efficient generation of conditional ROSA26-based mouse models that recapitulate oncogene activation in T-cell acute lymphoblastic leukemia

Abstract T-cell acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors that result from the malignant transformation of T-cell progenitors. Due to intensified chemotherapy, the prognosis of T-ALL has gradually improved. Nevertheless, this clinical improvement is most pronounced in pediatric treatment protocols, whereas adult patients more often present with primary resistant or relapsed disease. Our aim is to develop new mouse models that mimic oncogenic lesions identified in human disease. These mouse models are critically required to further enhance our knowledge on the molecular mechanisms that drive T-cell leukemogenesis. Moreover, they serve as important preclinical models to evaluate new therapeutic strategies for the treatment of human T-ALL. Here, we generated a fast method for embryonic stem cell (ESC) targeting and mouse chimera production. We constructed gateway-compatible vectors that allow tailor-made targeting vector design, including conditional expression of a transgene combined with an eGFP/luciferase reporter from the ROSA26(R26)-promoter. The final targeting vector is recombination-mediated cassette exchange (RMCE)-compatible and can be inserted in a genetically engineered R26-locus via RMCE. Correct integration of the incoming construct reactivates the NeoR gene and results in up to 100% ESC targeting efficiencies. Using our technology, we generated R26-based conditional knock-in mouse models for putative oncogenes that have previously been implicated in T-ALL disease biology. More specifically, the MYB leucine zipper transcription factor is aberrantly activated in a subset of T-ALL patients through T-cell receptor driven translocations (t(6;7)(q23;q34)) or genomic duplications of the MYB locus itself. To study the in vivo roles of cMyb in the pathogenesis of T-ALL, we used the above-mentioned genomic engineering technology to generate cMyb conditional knock-in mouse models. These mice were crossed with VaviCre mice to obtain hematopoietic specific expression of Myb and the EGFP/luciferase from the ROSA26-promoter. We demonstrated that forced expression of Myb in hematopoietic precursors is sufficient to drive T-cell leukemia in mice. To allow comparative analysis of tumors with and without T-cell specific Myb expression, we also genetically deleted the tumor suppressor Pten in these mice and found that cMyb expression synergizes with Pten deletion and resulted in fully penetrant and accelerated T-ALL formation. These data demonstrate an important role for Myb in the pathologenesis of T-ALL. Finally, we use this novel murine T-ALL model to find new therapies for T-ALL. Importantly, the tumor cells from the cMyb knock-in mice are luciferase positive and are therefore suitable for in vivo drug testing using bioluminescence. Because cMYB activation drives an oncogenic transcription program by regulation of super-enhancer activity in human T-ALL will test drugs that impede Myb protein stability or Myb-mediated transactivation. Citation Format: Tim Pieters, Sara TSas, Beatrice Lintermans, Sofie Peirs, Filip Mathijssens, Jody Haigh, Geert Berx, Steven Goossens, Pieter Van Vlierberghe. Fast and efficient generation of conditional ROSA26-based mouse models that recapitulate oncogene activation in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A08.

Abstract A07: Speedy mouse models to study melanomagenesis

Abstract Melanoma accounts for only about 1% of the skin cancer, but contributes to a vast majority of skin cancer deaths. Despite extensive progress in treating melanoma with immunotherapy and small-molecule inhibitors, melanoma patients still suffer from tumor recurrence, mainly due to inherent or acquired resistance to these treatments. A better understanding of the detailed molecular mechanisms that contribute to melanoma formation and resistance may provide novel avenues for the development of improved treatment strategies. Genetically engineered mouse models (GEMMs) closely recapitulate the genetics and etiology of human malignancies and have significantly contributed to our understanding of the mechanisms underlying melanoma initiation and progression. However, the generation and characterization of GEMMs is often challenging due to their time-consuming, laborious, and expensive nature. To overcome some of these limitations, we sought to develop a melanoma mouse-modeling platform, which allows for accelerated speed, increased throughput, and lower cost of in vivo melanoma studies compared to conventional mouse models. To this end, we established several embryonic stem cell (ESC) lines harboring multiple alleles to aide in creating versatile melanoma mouse models. These alleles include Cre-inducible endogenous BrafV600E or NrasQ61R alleles for melanoma initiation, conditional PTEN knockout alleles for melanoma progression, the melanocyte-specific, tamoxifen-inducible Tyr-CreERt2 allele, a Cags-LSL-rtTA3 allele to activate Tet-inducible expression cassettes, and the collagen homing cassette (CHC) for the efficient delivery of such expression cassettes via recombination-mediated cassette exchange (RMCE). We demonstrated that our newly derived ESCs can be targeted with various constructs via RMCE with extremely high efficiency, thus significantly reducing the number of clones one has to screen for successful targeting. Moreover, we confirmed pluripotency of our ESC lines by Oct4 and Nanog staining, and by demonstrating their ability to generate chimeras via blastocyst injection and completely ESC-derived mice via tetraploid complementation. Topical application of 4-hydroxytamoxifen (4-OHT) on chimeras carrying the BrafV600E and PTENFlox alleles resulted in the development of nevi and melanomas within a few weeks. Importantly, our melanoma ESC-GEMM approach is compatible with novel genetic tools including inducible shRNA and CRISPR, thus providing a versatile and modular platform for the study of gene function in melanomagenesis. Thus, we have established a speedy mouse-modeling platform that will stimulate and accelerate in vivo melanoma research, and will provide a useful resource to investigate the pathobiology of melanomagenesis and to develop and preclinically test novel treatments. Citation Format: Ilah Bok, Florian A. Karreth. Speedy mouse models to study melanomagenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A07.

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically Encoded Multiplexed Reporter Assay in Escherichia coli.

Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In Escherichia coli, decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our data set using a simple log-linear statistical model. Simple neural network models explain >95% of the variance in our data set by capturing nonlinear interactions with the spacer, background, and UP elements.

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies.

Site-specific recombinases (SSRs) such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange.

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.

Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

Abstract 2429: Temporally and spatially inducible mouse model of aneuploidy

Abstract Aneuploidy, the state in which cells have an abnormal number of chromosomes, is a hallmark of most human cancers. Despite aneuploidy's near-ubiquitous presence in tumors, it is unknown whether aneuploidy is a driver or passenger in cancer progression. Aneuploidy is normally prevented by the spindle assembly checkpoint (SAC), a critical cellular mechanism which blocks chromosomal missegregation by inhibiting premature sister chromatid separation during mitosis. Although transgenic mouse models with defective SAC proteins exist, the dysfunctional proteins are expressed constitutively from birth in all tissues, a milieu that differs from that of an in vivo human tumor in which cancer cells develop aneuploidy de novo in the context of a euploid organism. In order to generate a mouse model that more closely approximates this tumor environment, we will insert single copies of shRNA hairpins that target SAC proteins BubR1 or Mad2 into the mouse genome at the Col1A1 locus using recombination mediated cassette exchange. shRNA expression will be under the control of a tetracycline responsive element in tissues that co-express rtTA, leading to temporally and spatially inducible disruption of the SAC, and thus aneuploidy. In vitro shRNA construct validation experiments indicate our shRNAs successfully knockdown target RNA and protein levels, as well as functionally promote mitotic slippage after nocodazole arrest, which is indicative of an impaired SAC. We will use this mouse model to investigate the role of increased chromosomal missegregation on tumor initiation and evolution, as well as the impact of increased aneuploidy on normal organ function. [Daniel Eichberg is a Howard Hughes Medical Institute Medical Research Fellow.] Citation Format: Daniel Eichberg, Kristin Knouse, Angelika Amon. Temporally and spatially inducible mouse model of aneuploidy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2014-2429

Noncoding RNAs of the Ultrabithorax Domain of the Drosophila Bithorax Complex

RNA transcripts without obvious coding potential are widespread in many creatures, including the fruit fly, Drosophila melanogaster. Several noncoding RNAs have been identified within the Drosophila bithorax complex. These first appear in blastoderm stage embryos, and their expression patterns indicate that they are transcribed only from active domains of the bithorax complex. It has been suggested that these noncoding RNAs have a role in establishing active domains, perhaps by setting the state of Polycomb Response Elements A comprehensive survey across the proximal half of the bithorax complex has now revealed nine distinct noncoding RNA transcripts, including four within the Ultrabithorax transcription unit. At the blastoderm stage, the noncoding transcripts collectively span ∼75% of the 135 kb surveyed. Recombination-mediated cassette exchange was used to invert the promoter of one of the noncoding RNAs, a 23-kb transcript from the bxd domain of the bithorax complex. The resulting animals fail to make the normal bxd noncoding RNA and show no transcription across the bxd Polycomb Response Element in early embryos. The mutant flies look normal; the regulation of the bxd domain appears unaffected. Thus, the bxd noncoding RNA has no apparent function.

A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.