MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed-batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics.bispecific antibody, evolved host, hydrogen peroxide, redox | INTRODUCTIONRecently, an interest in the cellular redox state and its effects on recombinant protein production has emerged (Handlogten et al., 2017(Handlogten et al., , 2020Orellana et al., 2015). Reactive oxygen species (ROS) are partial reduction products of molecular oxygen generated as a result of mitochondrial oxidative phosphorylation and oxidative protein folding within the endoplasmic reticulum (ER; Chevallier

Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors. Video LinkThe video component of this article can be found at http://www.jove.com/video/52155/ 15 . The recombination potential is conferred by inducible expression of the Red recombination proteins of the phage 16,17 or the RecET proteins of the rac prophage 18. The Red/RecE exonuclease converts linear dsDNA to a single-stranded

Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.

Highlights 19(1) Constitutively active Ras1.GV prolongs Cdc42 activation in S. pombe pheromone signalling 20(2) Ras1.GV results in an immediate but only transient MAPK Spk1 activation 21(3) The RAS effector pathways MAPK Spk1 and Cdc42 compete with each other for active Ras1 22 (4) Predictive modelling explains MAPK Spk1 activation dynamics in 24 signaling-mutants 23 24 eTOC Blurb 25 S. pombe Ras1 activates the MAPK Spk1 and Cdc42 pathways. Kelsall et al. report that the 26 constitutively active Ras1.G17V mutation, which causes morphological anomalies, induces 27 prolonged Cdc42 activation but only a transient MAPK Spk1 activation followed by attenuation. 28 Mathematical modelling and biochemical data suggest a competition between the MAPK Spk1 and 29 Cdc42 pathways for active Ras1. 30 31 Summary 32 33 The small GTPase RAS is a signalling hub for many pathways and oncogenic human RAS 34 mutations are assumed to over-activate all of its downstream pathways. We tested this 35 assumption in fission yeast, where, RAS-mediated pheromone signalling (PS) activates the 36 MAPK Spk1 and Cdc42 pathways. Unexpectedly, we found that constitutively active Ras1.G17V 37 induced immediate but only transient MAPK Spk1 activation, whilst Cdc42 activation persisted. 38 Immediate but transient MAPK Spk1 activation was also seen in the deletion mutant of Cdc42-39 GEF Scd1 , a Cdc42 activator. We built a mathematical model using PS negative-feedback circuits 40 and competition between the two Ras1 effectors, MAPKKK Byr2 and Cdc42-GEF Scd1 . The model 41 robustly predicted the MAPK Spk1 activation dynamics of an additional 21 PS mutants. Supporting 42 the model, we showed that a recombinant Cdc42-GEF Scd1 fragment competes with MAPKKK Byr2 43 for Ras1 binding. Our study has established a concept that the constitutively active RAS 44 propagates differently to downstream pathways where the system prevents MAPK 45 overactivation. 46 47 Key words 48 Ras, MAPK, Cdc42/Rac, yeast pheromone signalling 49 50 Ras1 also regulates cell morphology during vegetative growth; whilst deletion of either gpa1, 83 MAPKKK byr2 , MAPKK byr1 or MAPK spk1 does not result in any obvious phenotypes during vegetative 84 cell growth (Obara et al., 1991;Sipiczki, 1988;Toda et al., 1991), ras1 cells lose the typical rod-85 shape morphology of fission yeast to become rounded (Fukui et al., 1989; Nadin-Davis et al., 86 1986a). Studies based on recombinant protein assays and yeast-2-hybrid analysis demonstrated 87 that Ras1 interacts with both MAPKKK Byr2 and Scd1, a GDP-GTP exchange factor (GEF) for Cdc42, 88 which regulates the actin cytoskeleton and cell morphology (Chang et al., 1994; Gronwald et al., 89 2001;Tu et al., 1997). These observations suggest that Ras1 simultaneously regulates both the 90 pheromone MAPK Spk1 and the Cdc42 pathways at the cell membrane (Weston et al., 2013). 91Indeed, a dynamic "Cdc42 zone" at the cell cortex prior to mating has been observed (Merlini et 92 al., 2013) and Ras1 and MAPK Spk1 cascade components are found there ...

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