Cassette Exchange Research Articles

A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO Cells.

As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO-S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837-1846. © 2017 Wiley Periodicals, Inc.

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

A Method for Validating Mutations Associated with Malignant Hyperthermia using CRISPR/Cas9 and Dual Integrase Cassette Exchange

Malignant Hyperthermia (MH) is a pharmacogenetic disorder characterized by a hypermetabolic skeletal muscle response to volatile anesthetics. While there are hundreds of mutations in the Ryanodine Receptor 1 gene (RyR1) associated with MH, the current diagnostic protocol has only confirmed 35 mutations to be causative. This study aims to develop a more efficient method for validating mutations. The CRISPR/Cas9 system has emerged as an efficient and targetable gene-editing tool used to assist in homology-driven repair (HDR) for the insertion of donor templates in vitro. Serine integrase site-specific recombination offers a more efficient method of gene editing but requires specific recognition sites. In this study, we use CRISPR/Cas9 and Dual Integrase Cassette Exchange (DICE) to create RyR1-mutant cell lines. Using calcium imaging, myotubes consisting of differentiated myoblasts are tested for their sensitivity to the calcium-releasing triggers KCl, caffeine, and 4-chloro-m-cresol. The sensitivities of RyR1-mutant myotubes are compared to wild-type myotubes. An increase in sensitivity is indicative of MH and is used to classify the mutations as likely causative. The study is performed on primary murine myoblasts that have been immortalized via lentiviral transduction of cyclin-dependent kinase 4 (CDK4). We have designed and tested guide RNAs (gRNAs) used in the CRISPR/Cas9 system, identifying three gRNAs with DNA-cleavage efficiencies of 25-30%. The combined use of CRISPR/Cas9 and DICE allows for the efficient and rapid creation of RyR1-mutant cell lines in which the mutations can subsequently be functionally validated. In doing so, we hope to expedite the validation of MH-associated mutations for the purpose of assisting the development of a genetic screen. In addition, our methods can be applied to other regions of the genome to create an improved, high-throughput method of genomic engineering.

Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.

The site‐specific excision of a target DNA sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. Currently, site‐specific recombinases (SSRs), such as Cre or Flp recombination target cassettes, have been successfully excised or inverted by a single SSR to regulate transgene expression. However, the use of a single SSR might restrict the complex control of gene expression. This study investigated the potential for expanding the multiple regulation of transgenes using three different integrase systems (TP901‐1, R4, and Bxb1). We designed three excision cassettes that expressed luciferase, where the luciferase expression could be exchanged to a fluorescent protein by site‐specific recombination. Individual cassettes that could be regulated independently by a different integrase were connected in tandem and inserted into a mouse artificial chromosome (MAC) vector in Chinese hamster ovary cells. The transient expression of an integrase caused the targeted luciferase activity to be lost and fluorescence was activated. Additionally, the integrase system enabled the specific excision of targeted DNA sequences without cross‐reaction with the other recombination targets. These results suggest that the combined use of these integrase systems in a defined locus on a MAC vector permits the multiple regulation of transgene expression and might contribute to genomic or cell engineering.

Use of the DICE (Dual Integrase Cassette Exchange) System.

When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone. When a single recombinase is used for RMCE, recombination between undesired pairings of the sites can lead to a significant number of unwanted cell lines. To reduce the frequency with which these side products occur, several variants of RMCE that increase desired outcomes have been developed. Nevertheless, an important feature lacking from these improved RMCE methods is that none have fully utilized the recombinases that have been demonstrated to be the most robust and stringent at performing genomic integration in plants and animals, i.e., the phiC31 and Bxb1 phage integrases. To address this need, we have developed an RMCE protocol using these two serine integrases that we call dual integrase cassette exchange (DICE). Our DICE system provides a means to achieve high-precision DNA integration at a desired location and is especially well suited for repeated recombination into the same locus. In this chapter, we provide our most current protocols for using DICE in feeder-free human-induced pluripotent stem cells .

Bxb1 phage recombinase assists genome engineering in Drosophila melanogaster.

Rapid and reliable genome modifications provide the basis for detailed in vivo functional analysis of any genomic entity (gene, regulatory DNA, non-coding RNA, etc). With the advent of CRISPR/Cas9 genome editing technology, manipulation of a particular genomic locus has become a routine undertaking in variety of model organisms, including the fruit fly Drosophila melanogaster. To further diversify the available tools for genome engineering, we successfully harnessed the phage recombinase Bxb1 to perform recombinase-mediated cassette exchange (RMCE) in D. melanogaster. We demonstrate that Bxb1 possesses highly efficient recombinase activity and could be used alone or in conjunction with other currently available recombinases for creating platforms for cassette exchange of targeted loci.

Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

BioSpotlight / Citations

BioTechniquesVol. 62, No. 1 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114489AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Scoring bacterial tumbles and runsMethods to study bacterial motility range from soft agar plate assays to microscopic tracking of bacterial movement. The latter method is much more precise and quantitative than the former, but it is also costlier and more complex due to need for high speed optical equipment, large amounts of data storage, and complex computation. In this month's issue of BioTechniques, William Bentley and his colleagues at the University of Maryland, College Park, describe TumbleScore, a MATLAB-based, freely available software for rapid tracking and quantification of bacteria swimming from movies with slow or fast frame-rates obtained from light or fluorescence microscopes. After the paths of the bacteria have been determined, software tools can be used to quantify complex motility behavior. For flagellated bacteria, the software provides various metrics for the phenomenon of tumbling, where the smooth path of the bacterium during a run propelled by the normal counterclockwise motion of the flagellum is interrupted by a sudden and random change in direction caused by a brief clockwise flagellar motion.See “TumbleScore: Run and tumble analysis for low frame-rate motility videos” on page 31.Another recombinase for rmceSite-specific recombinases such as Flp, Cre, and FC31 have been successfully exploited by researchers to rapidly and reliably modify the Drosophila melanogaster genome. One particularly useful application of site-specific recombinases is recombinase-mediated cassette exchange (RMCE), in which an acceptor cassette containing a transgenic construct flanked by sites specific for a recombinase is introduced into the Drosophila genome at a specific locus. In the presence of the site-specific recombinase, this acceptor cassette can be readily exchanged with a donor cassette containing a different transgenic construct flanked by the same recombinase-specific sites. This allows easy sequential introduction of any number of different cassettes into that specific genomic location. In this month's issue, Roumen Voutev and Richard S. Mann at Columbia University have adapted RMCE to work with the newer site-specific phage recombinase Bxb1. They demonstrate that Bxb1 can be efficiently used for RMCE, either alone or in parallel with other recombinases already in use for RMCE, further extending the versatility of RMCE in Drosophila.See “Bxb1 phage recombinase assists genome engineering in Drosophila melanogaster” on page 37.Faster tissue clearing chipsThe ability to image deep inside tissues is important for basic biological research as well as for drug discovery. Methods such as CLARITY have made it possible to render opaque tissues transparent for deep imaging, but these clearing protocols are very time-consuming. Chen et al. now report a microfluidic chip–based strategy to clarify 3-D tissue samples for structural analysis that is 20 times faster than other approaches and also amenable to high-throughput screening. The key to this method lies in the 567-fold increase in the rate of exchange of interstitial fluids that can be achieved on the chip, resulting in a more rapid removal of light-scattering lipids from tissue samples. The authors used their new system to image subpopulations of cells during tumor spheroid formation and to monitor vascular degradation in murine pancreatic islets over time. This new microfluidic workflow should greatly enhance organ-on-a-chip studies as well as high-throughput drug screening efforts.Chen et al. 2016. “Clarifying intact 3D tissues on a microfluidic chip for high-throughput structural analysis” Proc. Natl. Acad. Sci. [Epub ahead of print] doi:10.1073/pnas.1609569114Test-driving nanopore sequencingOxford Nanopore Technologies' MinION platform is the first nanopore-based single-molecule nucleic acid sequencing system to be released commercially. As more researchers begin to use nanopore sequencing, it's important that they understand the capabilities of this new technology. Oikonomopoulos et al. provide one such benchmark by testing the performance of the MinION platform for quantitative and qualitative assessment of cDNA populations. The authors sequenced a defined set of polyadenylated transcripts as well as a population of cDNAs from an HEK-293 cell line using the MinION platform, the Illumina HiSeq2500, and the PacBio RS II. Their MinION data were in good agreement with that of the other two most established platforms when it came to measuring cDNA abundance and length. The authors conclude that the MinION system can be used for reliable quantification of either long or short cDNA molecules in complex samples.Oikonomopoulos et al. 2016. “Benchmarking of the Oxford Nanopore MinION sequencing for quantitative assessment of cDNA populations” Scientific Reports 6:31602. doi:10.1038/srep31602FiguresReferencesRelatedDetails Vol. 62, No. 1 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print January 2017 Information© 2017 Author(s)PDF download

Dual Recombinase-Mediated Cassette Exchange by Tyrosine Site-Specific Recombinases.

Recombinase-mediated cassette exchange, or RMCE, is a genome engineering tool that can be used to swap DNA fragments of interest between two DNA molecules. In a variation of RMCE, called dual RMCE, the exchange of DNA fragments is mediated by two recombinases in contrast to one recombinase in the classic RMCE reaction. Under optimal conditions, the efficiency of dual RMCE can be quite high: up to ~45% of the transfected cells depending on the recombinase pair used to mediate the replacement reaction. Here we describe protocols for preparing for, performing, and optimizing the parameters of dual RMCE.

Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified.

B Cell-Based Seamless Engineering of Antibody Fc Domains.

Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the <em>AAVS1</em> Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the <em>AAVS1</em> Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

A trait stacking system via intra-genomic homologous recombination

A gene targeting method has been developed, which allows the conversion of 'breeding stacks', containing unlinked transgenes into a 'molecular stack' and thereby circumventing the breeding challenges associated with transgene segregation. A gene targeting method has been developed for converting two unlinked trait loci into a single locus transgene stack. The method utilizes intra-genomic homologous recombination (IGHR) between stably integrated target and donor loci which share sequence homology and nuclease cleavage sites whereby the donor contains a promoterless herbicide resistance transgene. Upon crossing with a zinc finger nuclease (ZFN)-expressing plant, double-strand breaks (DSB) are created in both the stably integrated target and donor loci. DSBs flanking the donor locus result in intra-genomic mobilization of a promoterless selectable marker-containing donor sequence, which can be utilized as a template for homology-directed repair of a concomitant DSB at the target locus resulting in a functional selectable marker via nuclease-mediated cassette exchange (NMCE). The method was successfully demonstrated in maize using a glyphosate tolerance gene as a donor whereby up to 3.3% of the resulting progeny embryos cultured on selection medium regenerated plants with the donor sequence integrated into the target locus. The process could be extended to multiple cycles of trait stacking by virtue of a unique intron sequence homology for NMCE between the target and the donor loci. This is the first report that describes NMCE via IGHR, thereby enabling trait stacking using conventional crossing.

Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System.

Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.

Tetracycline Selective Pressure and Homologous Recombination Shape the Evolution of Chlamydia suis: A Recently Identified Zoonotic Pathogen.

Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct. Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

Abstract 1348: A novel bicistronic reporter-based high-throughput screening platform for discovery of transcription-targeted lead compounds

Abstract Given that aberrant gene expression is widely spread in cancer, there is an imperative need of developing reliable high-throughput drug screening (HTS) platforms in search of lead compounds that can rectify abnormal expression of cancer-driving genes for treating this deadly disease. Although it has been demonstrated that reporter gene assays are useful in the identification of transcription-targeted lead compounds, these assays are often unreliable and can yield high rates of false compounds due to the facts that reporter genes are driven by cloned promoters and localized in foreign chromatin environments. While cloned promoters lack essential cis-regulatory elements (e.g., enhancers) far removed from transcription start sites, foreign chromatin environment can alter gene expression in a way atypical to endogenous genes. To address these limitations, we employed emerging genome-editing tools to develop a novel reporter-based HTS assay whereby the reporter gene is inserted immediately downstream of the coding region of an endogenous gene, and thereby co-expressed bicistronically with the endogenous gene as a single transcript under the control of the native transcriptional regulatory machinery. As a proof-of-principle, we first employed the CRISPR-Cas9 system to knock a fused selection gene tk-ble (encoding Zeocin resistance for selection for targeting events and thymidine kinase for negative selection for the removal of the selection gene, respectively) flanked by a wild-type and a mutated FRT fragment into the eukaryotic translation initiation factor 4E (EIF4E) gene locus, and then replaced the selection gene with a firefly luciferase (FLuc), or a Renilla luciferase (RLuc), by cassette exchange mediated by the recombinase Flp. We demonstrated that the FLuc gene was expressed as single transcripts with EIF4E by Northern blotting, and responded to chemical treatments in a way analog to the endogenous EIF4E gene. Using the cells carrying bicistronic FLuc reporter, we screened a chemical library containing 4,800 compounds for small molecules inhibitory for EIF4E expression, and identified 11 hits, out of which 6 compounds were validated to decrease EIF4E expression by qRT-PCR. While the 5 false-positive compounds were likely yielded due to their direct inhibition of FLuc activity, 4 of these false positives could be readily identified by an orthogonal assay using recombinant cells carrying RLuc in the same EIF4E locus. Therefore, our results have demonstrated that screening assays based on bicistronic in-situ reporters are more reliable, and powerful in searching for transcription-targeted lead compounds with high confidence. Citation Format: Liwei Lang, Chunhong Yan. A novel bicistronic reporter-based high-throughput screening platform for discovery of transcription-targeted lead compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1348.

Identifying Products of Recombinase-Mediated Cassette Exchange (RMCE) in Schizosaccharomyces pombe.

Homologous recombination is highly efficient when mediated between two identical target sequences by recombination enzymes such as Cre. Exploiting this, recombinase-mediated cassette exchange (RMCE) was developed for the genetic manipulation of eukaryotic cells, including those of Schizosaccharomyces pombe RMCE can be summarized in three stages: (1) A loxP-ura4(+)-loxM3 cassette is introduced into the genome using standard homologous recombination techniques to create a "base strain." (2) A Cre-expression plasmid carrying a protein tag or replacement gene flanked by loxP and loxM3 is introduced into the cell. (3) Cassette exchange between the chromosomal cassette and the plasmid cassette results in either gene tagging or gene replacement. This is selected for by loss of the marker. This protocol explains how to identify the products of the exchange events in the last stage.

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

ABSTRACTChinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.