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.

Dynamic rewiring of the Drosophila retinal determination network switches its function from selector to differentiation.

Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the "retinal determination gene network," composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development.

Expanding the Drosophila toolkit for dual control of gene expression.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA-system or QF-system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterizsed GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue-specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

Expanding the Drosophila toolkit for dual control of gene expression.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

In order for researchers to understand how organisms develop and function, they often switch specific genes on or off in certain tissues or at selected times. This can be achieved using genetic tools called binary expression systems. In the fruit fly ­ a popular organism for studying biological processes ­ the most common is the GAL4/UAS system. In this system, a protein called GAL4 is expressed in a specific organ or tissue where it activates a UAS element ­ a genetic sequence that is inserted in front of the gene that is to be switched on. This can also include genes inserted into the fruit fly encoding fluorescent proteins or stretches of DNA coding for factors that can silence specific genes. For example, fruit flies expressing GAL4 protein specifically in nerve cells and a UAS element in front of a gene for a fluorescent protein will display fluorescent nerve cells, which can then be examined using fluorescence microscopy. Studying how organs communicate with one other can require controlled expression of multiple genes at the same time. In fruit flies, other binary expression systems that are analogous to the GAL4/UAS system (known as LexA/LexAop and QF/QUAS) can be used in tandem. For example, to study gut-brain communication, the GAL4/UAS system might be used to switch on the gene for an insulin-like protein in the gut, with one of the other systems controlling the expression of its corresponding receptor in the brain. However, these experiments are currently difficult because, while there are thousands of GAL4/UAS genetic lines, there are only a few LexA/LexAop and QF/QUAS genetic lines. To address this lack of resources, Zirin et al. produced a range of genetically engineered fruit flies containing the LexA/LexAop and QF/QUAS binary expression systems. The flies expressed LexA or QF in each of the major fly organs, including the brain, heart, muscles, and gut. A fluorescent reporter gene linked to the LexAop or QUAS elements, respectively, was then used to test the specificity to single organs and compare the different systems. In some organs the LexA/LexAop system was more reliable than the QF/QUAS system. However, both systems could be successfully combined with genetic elements to switch on a fluorescent reporter gene or switch off a gene of interest in the intended organ. The resources developed by Zirin et al. expand the toolkit for studying fruit fly biology. In future, it will be important to understand the differences between GAL4, LexA and QF systems, and to increase the number of fruit fly lines containing the newer binary expression systems.

Control of Gliotactin localization and levels by tyrosine phosphorylation and endocytosis is necessary for survival of polarized epithelia.

The tricellular junction (TCJ) forms at the convergence of bicellular junctions from three adjacent cells in polarized epithelia and is necessary for maintaining the transepithelial barrier. In the fruitfly Drosophila, the TCJ is generated at the meeting point of bicellular septate junctions. Gliotactin was the first identified component of the TCJ and is necessary for TCJ and septate junction development. Gliotactin is a member of the neuroligin family and associates with the PDZ protein discs large. Beyond this interaction, little is known about the mechanisms underlying Gliotactin localization and function at the TCJ. In this study, we show that Gliotactin is phosphorylated at conserved tyrosine residues, a process necessary for endocytosis and targeting to late endosomes and lysosomes for degradation. Regulation of Gliotactin levels through phosphorylation and endocytosis is necessary as overexpression results in displacement of Gliotactin away from the TCJ throughout the septate junction domain. Excessive Gliotactin in polarized epithelia leads to delamination, paired with subsequent migration, and apoptosis. The apoptosis and the resulting compensatory proliferation resulting from high levels of Gliotactin are mediated by the Drosophila JNK pathway. Therefore, Gliotactin levels within the cell membrane are regulated to ensure correct protein localization and cell survival.

Engineering advanced cancer therapies with synthetic biology.

Engineered immune-cell-based cancer therapies have demonstrated robust efficacy in B cell malignancies, but challenges such as the lack of ideal targetable tumour antigens, tumour-mediated immunosuppression and severe toxicity still hinder their therapeutic efficacy and broad applicability. Synthetic biology can be used to overcome these challenges and create more robust, effective adaptive therapies that enable the specific targeting of cancer cells while sparing healthy cells. In this Progress article, we review recently developed gene circuit therapies for cancer using immune cells, nucleic acids and bacteria as chassis. We conclude by discussing outstanding challenges and future directions for realizing these gene circuit therapies in the clinic.

Comparison of Integrases Identifies Bxb1-GA Mutant as the Most Efficient Site-Specific Integrase System in Mammalian Cells.

Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells. Bxb1-GA point mutant sites do not cross-react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. In comparison, we test the efficiency and orthogonality of ϕC31 and Wß integrases, and show that Wß has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines.

Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology.

One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future.

Drosophila eyes absent is required for normal cone and pigment cell development.

In Drosophila, development of the compound eye is orchestrated by a network of highly conserved transcriptional regulators known as the retinal determination (RD) network. The retinal determination gene eyes absent (eya) is expressed in most cells within the developing eye field, from undifferentiated retinal progenitors to photoreceptor cells whose differentiation begins at the morphogenetic furrow (MF). Loss of eya expression leads to an early block in retinal development, making it impossible to study the role of eya expression during later steps of retinal differentiation. We have identified two new regulatory regions that control eya expression during retinal development. These two enhancers are necessary to maintain eya expression anterior to the MF (eya-IAM) and in photoreceptors (eya-PSE), respectively. We find that deleting these enhancers affects developmental events anterior to the MF as well as retinal differentiation posterior to the MF. In line with previous results, we find that reducing eya expression anterior to the MF affects several early steps during early retinal differentiation, including cell cycle arrest and expression of the proneural gene ato. Consistent with previous observations that suggest a role for eya in cell proliferation during early development we find that deletion of eya-IAM leads to a marked reduction in the size of the adult retinal field. On the other hand, deletion of eya-PSE leads to defects in cone and pigment cell development. In addition we find that eya expression is necessary to activate expression of the cone cell marker Cut and to regulate levels of the Hedgehog pathway effector Ci. In summary, our study uncovers novel aspects of eya-mediated regulation of eye development. The genetic tools generated in this study will allow for a detailed study of how the RD network regulates key steps in eye formation.

Regulation of Drosophila eye development by the transcription factor Sine oculis.

Homeodomain transcription factors of the Sine oculis (SIX) family direct multiple regulatory processes throughout the metazoans. Sine oculis (So) was first characterized in the fruit fly Drosophila melanogaster, where it is both necessary and sufficient for eye development, regulating cell survival, proliferation, and differentiation. Despite its key role in development, only a few direct targets of So have been described previously. In the current study, we aim to expand our knowledge of So-mediated transcriptional regulation in the developing Drosophila eye using ChIP-seq to map So binding regions throughout the genome. We find 7,566 So enriched regions (peaks), estimated to map to 5,952 genes. Using overlap between the So ChIP-seq peak set and genes that are differentially regulated in response to loss or gain of so, we identify putative direct targets of So. We find So binding enrichment in genes not previously known to be regulated by So, including genes that encode cell junction proteins and signaling pathway components. In addition, we analyze a subset of So-bound novel genes in the eye, and find eight genes that have previously uncharacterized eye phenotypes and may be novel direct targets of So. Our study presents a greatly expanded list of candidate So targets and serves as basis for future studies of So-mediated gene regulation in the eye.

Genome-wide DNA binding pattern of the homeodomain transcription factor Sine oculis (So) in the developing eye of Drosophila melanogaster.

The eye of the fruit fly Drosophila melanogaster provides a highly tractable genetic model system for the study of animal development, and many genes that regulate Drosophila eye formation have homologs implicated in human development and disease. Among these is the homeobox gene sine oculis (so), which encodes a homeodomain transcription factor (TF) that is both necessary for eye development and sufficient to reprogram a subset of cells outside the normal eye field toward an eye fate. We have performed a genome-wide analysis of So binding to DNA prepared from developing Drosophila eye tissue in order to identify candidate direct targets of So-mediated transcriptional regulation, as described in our recent article [1]. The data are available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE52943. Here we describe the methods, data analysis, and quality control of our So ChIP-seq dataset.

Eyes absent tyrosine phosphatase activity is not required for Drosophila development or survival.

Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.

MAPK target sites of eyes absent are not required for eye development or survival in Drosophila.

Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that plays an essential role in eye development and survival in Drosophila. Ectopic eye induction assays using cDNA transgenes have suggested that mitogen activated protein kinase (MAPK) activates Eya by phosphorylating it on two consensus target sites, S402 and S407, and that this activation potentiates the ability of Eya to drive eye formation. However, this mechanism has never been tested in normal eye development. In the current study, we generated a series of genomic rescue transgenes to investigate how loss- and gain-of-function mutations at these two MAPK target sites within Eya affect Drosophila survival and normal eye formation: eya(+)GR, the wild-type control; eya(SA)GR, which lacks phosphorylation at the two target residues; and eya(SDE)GR, which contains phosphomimetic amino acids at the same two residues. Contrary to the previous studies in ectopic eye development, all eya genomic transgenes tested rescue both eye formation and survival equally effectively. We conclude that, in contrast to ectopic eye formation, MAPK-mediated phosphorylation of Eya on S402 and S407 does not play a role in normal development. This is the first study in Drosophila to evaluate the difference in outcomes between genomic rescue and ectopic cDNA-based overexpression of the same gene. These findings indicate similar genomic rescue strategies may prove useful for re-evaluating other long-standing Drosophila developmental models.