Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site.

The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

All-in-one IQ toggle switches with high versatilities for fine-tuning of transgene expression in mammalian cells and tissues.

The transgene toggling device is recognized as a powerful tool for gene- and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared with the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.

Targeted Integration of Transgenes at the Mouse Gt(ROSA)26Sor Locus.

The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes.Here we report a protocol for the generation of targeted transgenic alleles at Gt(ROSA)26Sor, taking as an example a conditional overexpression allele, by PhiC31 integrase/recombinase-mediated cassette exchange of an engineered Gt(ROSA)26Sor locus in mouse embryonic stem cells. The overexpression allele is initially silenced by the presence of a loxP flanked stop sequence but can be strongly activated through the action of Cre recombinase.

FRaeppli: a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish.

Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression.

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Site-specific integration as an efficient method for production of recombinant human hyaluronidase PH20 in semi-adherent cells.

PH20 is a hyaluronidase enzyme that can hydrolyze the glycosidic bond in hyaluronic acid as the major proteoglycan found in extracellular matrices. In the present study, we constructed and characterized two donor plasmids, one of them with one and the second with two PH20 expression cassettes. The expression vectors were site specifically integrated into the genome of HEK293T cells using PhiC31 integrase system to develop HEK293T stable cell lines secreting His-tagged recombinant human PH20 (rhPH20) in the culture supernatant. The produced rhPH20 was quantified using ELISA and turbidimetric assay tests, and its catalytic activity was also assessed by treating the mouse cumulus-oocyte complexes. Our results showed that the secreted rhPH20 in the culture supernatant had the specific activity of 16,660 IU/mg and the recombinant enzyme was able to remove the cumulus cells from oocytes. The results also indicated that phiC31 enzyme inserted the PH20-expressing donor vectors into the specific pseudo attP sites including 10q21.2 and 20q11.22 in the genome of the target cells with different copy numbers. Taken together, our findings demonstrate that PhiC31 integrase system is able to be applied as a robust tool for efficient production and secretion of soluble and active rhPH20 by HEK293T cells as a semi-adherent human cell line. KEY POINTS: • Efficient production of human recombinant PH20 in a semi-adherent human cell line • Successful application of PhiC31 integrase system for generation of stable recombinant clones • Use of a human cell line for expression of a recombinant human protein due to complex and efficient post-translational modifications and protein folding.

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.

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase.

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.

Heterogeneous expression of the ammonium transporter AgAmt in chemosensory appendages of the malaria vector, Anopheles gambiae.

Ammonia is one of the principal kairomones originating from human and other animal emanations and in that context, plays an essential role in the host-seeking behaviors of the malaria vector mosquito Anopheles gambiae. Nevertheless, despite its importance in directing host-seeking, the mechanisms underlying ammonia detection in the mosquito olfactory system remains largely unknown. In addition to ongoing efforts to identify and characterize the molecular receptors that underlie ammonia sensitivity, previous studies have revealed a prominent role for ammonium transporters (Amt) in modulating antennal and behavioral responses in Drosophila melanogaster and An. gambiae. In the former, localization of DmAmt in antennal sensilla to auxiliary cells surrounding the ammonia sensory neurons led to the hypothesis that its role was to clear excess ammonium ions in the sensillar lymph. In the latter, RT-PCR and heterologous expression have been used to examine the expression and functional characteristics of the An. gambiae ammonium transporter, AgAmt. We now employ advanced transgenic tools to comprehensively examine AgAmt spatial localization across the peripheral chemosensory appendages in larvae and adult female An. gambiae. In the larval antennae, AgAmt appears localized in both neuronal and auxiliary cells. In contrast to D. melanogaster, in the adult antennae, AgAmt-derived signals are observed in both non-neuronal auxiliary cells and in sensory neurons in ammonia-responsive basiconic and coeloconic sensilla. In the maxillary palps, labella, and tarsi, AgAmt appears restricted to sensory neurons. We have also characterized the responses to ammonia of adult antennal coeloconic sensilla and maxillary palp capitate pegs revealing a correlation between sensillar AgAmt expression and ammonia sensitivity. Taken together, these data suggest that AgAmt may play heterogeneous roles in the adult and larval chemosensory apparatus and potentially broad utility as a supra-receptor target in mosquito control.

Integrase-Mediated Targeted Transgenics Through Pronuclear Microinjection.

Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal and provides an extremely powerful tool to dissect the molecular mechanisms of disease. Transgenic mouse models made by microinjection of DNA into zygotic pronuclei, in particular, have been widely used by the genetics community for over 35 years. However, up till 5 years ago, it remained a rather crude approach: injected sequences randomly insert in multiple copies as concatemers, and they can be mutagenic and have variable, ectopic, or silenced expression depending on the site of integration, a phenomenon called position effects. As a result, multiple lines are required in order to confirm appropriate transgene expression. This can be partially overcome by flanking transgenes with insulator sequences to protect the transgene from influence of surrounding regulatory elements. Large (<300 kb) BAC-based transgenic vectors have also been shown to be more resistant to position effects. However, animals carrying extra copies of fairly large regions of the genome could have unpredictable phenotypes.These problems can be overcome by targeting the transgene to a specific chromosomal locus via homologous recombination in embryonic stem (ES) cells. However, this method is significantly more laborious and time consuming, as it involves creation of modified ES cells and mouse chimeras, as well as eventual germline transmission of the transgene.Here, I describe an integrase-based approach, trademarked as "TARGATT™" (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

A modular toolset of phiC31-based fluorescent protein tagging vectors for Drosophila.

The Drosophila transgenic technology and fluorescent protein fusions are powerful tools to analyze protein expression patterns, subcellular localization and protein dynamics. Recently, the Drosophila transgenic technology has been improved by the highly efficient phiC31 site-specific integration system. Many new and improved fluorescent proteins with desirable advantages have been developed. However, the phiC31 system and the newly developed fluorescent proteins have not been systematically applied in Drosophila transgenic vectors. Here, we have constructed a modular toolset of C-terminal fluorescent protein fusion vectors based on phiC31 site-specific integration system for the generation of transgenic Drosophila lines. These cloning vectors contain a variety of fluorescent tags, including blue, cyan, green or red fluorescent proteins, photoactivatable or photoswitchable fluorescent proteins, fluorescent timers, photosensitizers and bimolecular fluorescence complementation tags. These vectors provide a range of transcriptional regulation options including UAST, UASP, UASC, LexAop, QUAS, Ubi, αTub67C and αTub84B promoters, and two screening marker options including white and vermilion gene. The vectors have been tested in vivo and can produce fluorescent chimeric proteins that are functional.

Serine integrase recombinational engineering (SIRE): A versatile toolbox for genome editing.

Chromosomal integration of biosynthetic pathways for the biotechnological production of high-value chemicals is a necessity to develop industrial strains with a high long-term stability and a low production variability. However, the introduction of multiple transcription units into the microbial genome remains a difficult task. Despite recent advances, current methodologies are either laborious or efficiencies highly fluctuate depending on the length and the type of the construct. Here we present serine integrase recombinational engineering (SIRE), a novel methodology which combines the ease of recombinase-mediated cassette exchange (RMCE) with the selectivity of orthogonal att sites of the PhiC31 integrase. As a proof of concept, this toolbox is developed for Escherichia coli. Using SIRE we were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette. Next to integrating large fragments, the option for multitargeting, for deleting operons, as well as for performing in vivo assemblies further expand and proof the versatility of the SIRE toolbox for E. coli. Finally, the serine integrase PhiC31 was also applied in the yeast Saccharomyces cerevisiae as a marker recovery tool, indicating the potential and portability of this toolbox.

Construction of a Mammalian IRES-based Expression Vector to Amplify a Bispecific Antibody; Blinatumomab.

Blinatumomab, the bispecific T cell engager antibody (BsAb), has been demonstrated as the most successful BsAb to date. Throughout the past decade, vector design has great importance for the expression of monoclonal antibody in Chinese hamster ovary (CHO) cells. It has been indicated that expression vectors based on the elongation factor-1 alpha (EF-1 alpha) gene and DHFR selection marker can be highly effective to produce populations of stably transfected cells in the selection medium. Moreover, the phiC31 integrase system is considered as an attractive and safe protein expression system in mammalian cells and it could integrate a donor plasmid of any size, as a single copy, in to the host genome with no cofactors. In this study, phiC31 integrase technology in combination with DHFR amplification system was used to have an expression vector for future expression of blinatumomab in CHO cells. The gene of interest (BsAb gene) could be joined to DHFR selection marker with the insertion of an internal ribosome entry site (IRES). By positioning the DHFR downstream of BsAb gene and IRES, the transcription of the selection marker can depend on the successful transcription of the BsAb gene, which was located upstream in the expression construct. In this study, FC550A-1 vector was used as the backbone and DHFR selection marker was successfully combined with phiC31 integrase technology to generate a high-expressing construct for BsAb expression in CHO-DG44 cells in future studies.

Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice.

The discovery of new gene editing tools in the past several years has moved the transgenic field to a new level. The traditional random transgenesis method by pronuclear microinjection has been largely replaced by targeted or site-specific transgenic technologies without the need of homologous recombination in embryonic stem (ES) cells. In this chapter, I describe detailed protocols of an integrase-based approach, trademarked as "TARGATT™" (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into a predetermined chromosomal locus with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Parallel Genomic Engineering of Two Drosophila Genes Using Orthogonal attB/attP Sites.

Precise modification of sequences in the Drosophila melanogaster genome underlies the powerful capacity to study molecular structure-function relationships in this model species. The emergence of CRISPR/Cas9 tools in combination with recombinase systems such as the bacteriophage serine integrase ΦC31 has rendered Drosophila mutagenesis a straightforward enterprise for deleting, inserting and modifying genetic elements to study their functional relevance. However, while combined modifications of non-linked genetic elements can be easily constructed with these tools and classical genetics, the independent manipulation of linked genes through the established ΦC31-mediated transgenesis pipeline has not been feasible due to the limitation to one attB/attP site pair. Here we extend the repertoire of ΦC31 transgenesis by introducing a second pair of attB/attP targeting and transgenesis vectors that operate in parallel and independently of existing tools. We show that two syntenic orthologous genes, CG11318 and CG15556, located within a 25 kb region can be genomically engineered to harbor attPTT and attPCC sites. These landing pads can then independently receive transgenes through ΦC31-assisted integration and facilitate the manipulation and analysis of either gene in the same animal. These results expand the repertoire of site-specific genomic engineering in Drosophila while retaining the well established advantages and utility of the ΦC31 transgenesis system.

A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase.

Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (Oryzias latipes), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This system was pioneered in medaka using the Sleeping Beauty transposon system, and the attP site was established at three chromosomal locations. However, this number appeared insufficient with regard to genetic linkage between the attP-landing site and a genetically modified locus of interest. Here, to establish a collection of transgenic strains of medaka, we introduced an attP motif into the medaka genome using the Ac/Ds maize transposon system and established 12 independent transgenic strains harboring a single copy of the attP motif in at least 11 of the 24 medaka chromosomes. We designed an attB-targeting vector that was integrated efficiently and precisely into the attP-landing site, and with which the DNA of interest was efficiently transmitted to germline cells. Extraneous sequences in the integrants derived from the bacterial backbone of the attB-targeting vector as well as a transgenic fluorescence marker present in the attP-landing site were removable through flippase-mediated recombination. Further, an advanced targeting vector with a heart-specific recombination marker served as a useful tool for easily screening phiC31 integrase-mediated recombinant G0 embryos, leading to the efficient establishment of transgenic strains. Thus, our resources advance genetic research in medaka.

Rapid characterization of the CHO platform cell line and identification of pseudo attP sites for PhiC31 integrase.

The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In™ TI™ technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization. By applying these approaches, we were able to accurately identify the copy number of integrated R4-attP sites and the genomic position of recombination of many clones. Three novel PhiC31 pseudo-attP sites were identified on chromosomes 1, 3 and 6, and their genomic features were analyzed. The characterized platform cell lines are stable, and because of single-copy, site-specific R4 recognition attP site, the cell lines could be retargeted for recombinant protein production and drug discovery applications.

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 .

PhiC31 integrase can improve the efficiency of different construct designs for monoclonal antibody expression in CHO cells.

OBJECTIVES: Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. METHODS AND RESULTS: In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site. CHO-S cells were co-transfected with single ORF/IRES or dual ORF vectors along with a phiC31 integrase expression vector which can catalyze recombination between attB site and pseudo-attP sites presented in the mammalian genome. Our results demonstrated that dual ORF vector in the presence of phiC31 integrase expression vectors (+FC31 2P) generated more recombinant antibody in comparison to its negative control (-FC31 2P). Moreover, both of +FC31 2P and -FC31 2P cell pools yield higher recombinant protein in comparison to single ORF/IRES vector (FC31 IRES) cell pools. Stability of expression in phiC31 co-transfected cell pools (+FC31 2P and +FC31 IRES) had no considerable changes. CONCLUSIONS: Our results indicated that the dual ORF vector using integrase can support the generation of cell lines with stable transgene expression at an elevated mAb relative to single ORF/IRES vector.