Recombinant proteins used in biomedical research, diagnostics and different therapies are mostly produced in Chinese hamster ovary cells in the pharmaceutical industry. These biotherapeutics, monoclonal antibodies in particular, have shown remarkable market growth in the past few decades. The increasing demand for high amounts of biologics requires continuous optimization and improvement of production technologies. Research aims at discovering better means and methods for reaching higher volumetric capacity, while maintaining stable product quality. An increasing number of complex novel protein therapeutics, such as viral antigens, vaccines, bi- and tri-specific monoclonal antibodies, are currently entering industrial production pipelines. These biomolecules are, in many cases, difficult to express and require tailored product-specific solutions to improve their transient or stable production. All these requirements boost the development of more efficient expression optimization systems and high-throughput screening platforms to facilitate the design of product-specific cell line engineering and production strategies. In this minireview, we provide an overview on recent advances in CHO cell line development, targeted genome manipulation techniques, selection systems and screening methods currently used in recombinant protein production.Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Biotherapeutic glycoproteins have revolutionised the field of pharmaceuticals, with new discoveries and continuous improvements underpinning the rapid growth of this industry. N-glycosylation is a critical quality attribute of biotherapeutic glycoproteins that influences the efficacy, half-life and immunogenicity of these drugs. This review will focus on the advances and future directions of remodelling N-glycosylation in Chinese hamster ovary (CHO) cells, which are the workhorse of recombinant biotherapeutic production, with particular emphasis on antibody products, using strategies such as cell line and protein backbone engineering.© 2021 The Author(s).

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site.© 2017 Wiley Periodicals, Inc.

We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected.

Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae. To introduce double-strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence. Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis. Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them. Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.

Members of the Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.

Site-specific DNA recombinases can "cut and paste" DNA. For example, they can promote excision of specific DNA segments or insertion of new DNA segments in specific places. However, natural recombinases act only at their cognate recombination sites, so current applications are limited to genetically modified organisms in which these sites have been introduced into the genome. Transposases also catalyze DNA rearrangements; they promote insertion of specific DNA sequences but at nonspecific locations. Applicability of site-specific recombinases and transposases in experimental genetics, biotechnology, and gene therapy would be much wider if they could be re-engineered so as to act specifically at chosen sequences within an organism's natural genome. This review will discuss progress towards the creation of such "designer" recombinases.

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.

Engineered nucleases enable the targeted alteration of nearly any gene in a wide range of cell types and organisms. The newly-developed transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain that can be easily engineered so that TALENs can target essentially any sequence. The capability to quickly and efficiently alter genes using TALENs promises to have profound impacts on biological research and to yield potential therapeutic strategies for genetic diseases.

The availability of rapid and robust methods for controlling gene function is of prime importance not only for assigning functions to newly discovered genes, but also for therapeutic intervention. Traditionally, gene function has been probed by often-laborious methods that either increase the level of a gene product or decrease it. Advances now make it possible to rapidly produce zinc-finger proteins capable of recognizing virtually any 18 bp stretch of DNA--a sequence long enough to specify a unique address in any genome. The attachment of functional domains also allows the design of tailor-made transcription factors for specific genes. Recent studies demonstrate that artificial transcription factors are capable of controlling the expression of endogenous genes in their native chromosomal context with a high degree of specificity in both animals and plants. Dominant regulatory control of expression of any endogenous gene can be achieved rapidly and can be also placed under chemical control. A wide range of potential applications is now within reach.

Since its inception, the CRISPR/Cas9 technology has been widely utilized for the targeted insertion of donor DNAs into mammalian genomes. A shortcoming with the earlier knock-in (KI) approaches, however, has been the low efficiency of targeted integrations-especially in primary cells and mouse embryos. Since, a variety of novel strategies have been developed towards improving the KI efficiencies in select target cells. In this review, the current applications of CRISPR/Cas9-mediated KIs in mammalian cells are described. Furthermore, the recent strategies which have been developed in order to augment the CRISPR/Cas9-mediated KI efficiencies are summarized.Copyright © 2019 Elsevier B.V. All rights reserved.

Transposons are a group of mobile genetic elements that are defined as a DNA sequence. Transposons can jump into different places of the genome; for this reason, they are called jumping genes. However, some transposons are always kept at the insertion site in the genome. Most transposons are inactivated and as a result, cannot move. Transposons are divided into two main groups: retrotransposons (class І) and DNA transposons (class ІІ). Retrotransposons are often found in eukaryotes. DNA transposons can be found in both eukaryotes and prokaryotes. The bacterial transposons belong to the DNA transposons and the Tn family, which are usually the carrier of additional genes for antibiotic resistance. Transposons can transfer from a plasmid to other plasmids or from a DNA chromosome to plasmid and vice versa that cause the transmission of antibiotic resistance genes in bacteria. The treatment of bacterial infectious diseases is difficult because of existing antibiotic resistance that part of this antibiotic resistance is caused by transposons. Bacterial infectious diseases are responsible for the increasing rise in world mortality rate. In this review, transposons and their roles have been studied in bacterial antibiotic resistance, in detail.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.© 2019 Schertzer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

We have transferred the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination targets (FRTs), into the genome of Drosophila. Flies were transformed with an FLP gene under the control of hsp70 regulatory sequences and with a white gene flanked by FRTs. The heat-induced recombinase catalyzes recombination between FRTs, causing loss of white (seen somatically as white patches in the eye) and, less frequently, gain of white (seen as dark-red patches). Loss and gain frequencies vary with the severity of the heat shock, and patterns of mosaicism vary with the developmental stage at which the heat shock is applied. The recombinase is also active in the germline, producing white-eyed and dark-red-eyed progeny.

The R gene product (R protein) of Zygosaccharomyces rouxii plasmid pSR1 catalyzes site-specific recombination within a 58 base-pair (bp) sequence present in the 959 bp inverted repeats of this plasmid. The R protein was produced in Escherichia coli and partially purified. The partially purified protein catalyzed site-specific recombination in vitro without the supply of an energy source. Recombination resulted in intramolecular inversion or deletion, depending on whether the orientations of the two recombination sites on the substrate plasmid were the same or opposite. Presumably, R protein is the only protein required for the recombination reaction. A circular DNA molecule appears to be a better substrate than a linear molecule in R-mediated in vitro intramolecular recombination. The R protein binds to a set of six 12 bp elements within the inverted repeats of pSR1. Two of these 12 bp elements are arranged in an inverted configuration with a 7 bp spacer in the 58 bp sequence. The R protein mediates strand cleavage in vitro at the junction between the 12 bp elements and the 7 bp spacer. The cleavage sites on the top and bottom strands are staggered and flanked by polypurine tracts that form part of the 12 bp elements.

Molecular techniques now allow the design of precise genetic modifications in the mouse. Not only can defined nucleotide changes be engineered into the genome of the mouse, but genetic switches can be designed to target expression or ablation of any gene (for which basic molecular information is available) to any tissue at any defined time. These strategies promise to contribute substantially to an increased understanding of individual gene function in development and pathogenesis. A powerful tool, both for the design of such genetic switches and for speeding the creation of gene-modified animals, is the Cre site-specific DNA recombinase of bacteriophage P1. Precise DNA rearrangements and genetic switches can be efficiently generated in a straightforward manner using Cre recombinase. In conjunction with inducible systems for controlling Cre expression and function, these recombination-based strategies are likely to have a profound impact on developmental biology and the generation of useful animal models of human disease.

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, phi(w39) and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.

Evidence is presented that transcription of most of the early genes in the Streptomyces coelicolor A3(2) phage phi C31 is from a series of unusual promoters that depend on a function expressed early in the phi C31 lytic cycle. Primer extension analysis on the 5' ends of three early mRNAs, from samples prepared 10 min after induction of a thermosensitive phi C31 lysogen, showed that the 5' ends all mapped close to highly similar sequences, which are proposed to be an important part of phage-specific promoters. In a shotgun cloning experiment, a fragment containing one of these sequences strongly activated transcription of the xyIE reporter gene in plaques of a phi C31-derived promoter-probe vector. Another of the sequences was inserted into a xyIE-containing promoter-probe plasmid vector, and promoted xyIE expression only when the host was supporting the lytic cycle of phi C31. This suggested that a transcription factor needed for activity of the promoters was present only in phi C31-infected cells. Examination of published and unpublished phi C31 sequence data revealed several more sequences that closely resemble the conserved region of the characterized promoters. Most of these are found in positions close to apparent transcription start sites mapped previously by low-resolution S1 mapping. An overall consensus sequence for the conserved region suggests a general organization (though not a primary sequence) resembling that of promoters recognized in other bacteria by the sigma 54 form of RNA polymerase.

The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989). Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398-404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRI fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains, MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci.

Tyrosine-type site-specific recombinases (T-SSRs) have opened new avenues for the predictable modification of genomes as they enable precise genome editing in heterologous hosts. These enzymes are ubiquitous in eubacteria, prevalent in archaea and temperate phages, present in certain yeast strains, but barely found in higher eukaryotes. As tools they find increasing use for the generation and systematic modification of genomes in a plethora of organisms. If applied in host organisms, they enable precise DNA cleavage and ligation without the gain or loss of nucleotides. Criteria directing the choice of the most appropriate T-SSR system for genetic engineering include that, whenever possible, the recombinase should act independent of cofactors and that the target sequences should be long enough to be unique in a given genome. This review is focused on recent advancements in our mechanistic understanding of simple T-SSRs and their application in developmental and synthetic biology, as well as in biomedical research.

We describe the development of 'recCas9', an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A binary system for gene activation and site-specific integration, based on the conditional recombination of transfected sequences mediated by the FLP recombinase from yeast, was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequence to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporter. By the reverse reaction, integration of transfected DNA was targeted to a specific chromosomal site. The results suggest that FLP could be used to mosaically activate or inactivate transgenes for analysis of vertebrate development, and to efficiently integrate transfected DNA at predetermined chromosomal locations.

Stable transformants of mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We have eliminated both of these constraints on predictable gene expression by use of a lox recombination vector. The positive selection vector system is designed to directly select Cre-mediated DNA integration at a lox target previously placed into the genome of cultured mammalian cells. Proper targeting activates expression of a defective lox-neomycin phosphotransferase (neo) fusion gene target. With CHO cell lines containing this target, almost all of the selected transformants (54 of 56 independent G418-resistant colonies) were simple single-copy integrants of the targeting DNA. To monitor gene expression at a single chromosomal site, we used a beta-actin promoter-lacZ reporter construct. Independent G418-resistant colonies from site-specific integration of the reporter gene all showed nearly identical levels of beta-galactosidase activity when the reporter construct integrated at a particular chromosomal position. The same construct integrated at a second chromosomal position exhibited a slightly different level of activity, characteristic of that second position. These results show that Cre-mediated site-specific integration can facilitate the construction of isogenic cell lines and thereby permit reproducible gene expression in stably transformed cell lines.

Chinese hamster ovary (CHO) cell-based platforms are the most widely used for the biomanufacturing of complex therapeutic proteins, such as monoclonal antibodies (mAbs). The development of high-producing clones that are stable and amenable to large-scale cultures is essential to advance a molecule toward clinical evaluation. Nevertheless, the generation of such clones generally relies on random integration of an expression plasmid encoding the therapeutic protein gene into the host genome. The ensuing clone selection relying on empirical screens and cell line characterization is extensive and time-consuming. An emerging paradigm in CHO cell line development is the use of site-specific recombinases to enable the integration of therapeutic transgenes into pre-marked chromosomal locations with defined expression characteristics. Recombinase-mediated cassette exchange (RMCE) provides a sophisticated alternative to conventional CHO cell line development, leading to the generation of more consistent and reliable clones and may ultimately shorten the "time-to-clinic" of recombinant therapeutics. Herein, we review the recent advances in the use of site-specific recombination systems and their associated cassette exchange technologies for the rapid generation of stable CHO clones with predictable growth, stability, quality and productivity characteristics. Particular emphasis is placed on cassette exchange technologies currently used in the industry. We also discuss the technical hurdles associated with uses of site-specific recombinase systems in CHO cells, illustrate how these problems can be mitigated and provide a perspective on future work concerning these systems.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection.In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches.PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique.The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.Copyright© by Royan Institute. All rights reserved.

Conventional treatment for cancer such as surgical resection and chemotherapy can cause damage in cases with advanced cancers. Moreover, the identification of tumor-specific targets has great importance in T-cell therapies. For decades, T cell activity has been stimulated to improve anti-tumor activity. Bispecific antibodies have attracted strong interest from pharmaceutical companies, for their diagnostic and therapeutic use. Blinatumomab is a first-in-class bispecific T engager antibody for the treatment of relapsed or refractory precursor B- cell acute lymphoblastic leukemia. But, it can benefit several cases with CD19 malignancies in the future. PhiC31 integrase-based vectors could selectively integrate therapeutic transgenes into pseudo-attP sites in CHO genome. In this study, production of Blinatumomab in CHO cells using this type of vectors was investigated. We evaluated the effects of histone deacetylases (HDACs) inhibitors such as sodium butyrate and valproic acid, on specific productivity and cell viability of antibody expressing cells. Although sodium butyrate increased specific productivity about 1.7-fold and valproic acid about 1.4-fold, valproic acid was found more efficient because of its less cytotoxic effect on cell growth. We examined the efficacy of expressed Blinatumomab at various effector to target (E/T) ratios. A dose-response analyses of calcein-acetoxymethyl release assay illustrated that the effective dose of expressed mAb required for antibody mediated cytotoxicity was 100 ng/ml and the expressed mAb was more effective at E/T ratios of 10:1 and 5:1. Results of this study indicated that the expressed blinatumomab can be useful for enhancing the cytotoxicity of CD3 T-cells against CD19 target cells.

Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been the tool of choice to build long-term and stable synthetic memory in other organisms, because they enable a shift between two alternative states registering the information at the DNA level. Here we report a memory switch for whole plants based on the bacteriophage ϕC31 site-specific integrase. The switch was built as a modular device made of standard DNA parts, designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally operated by action of the ϕC31 integrase (Int), and its recombination directionality factor (RDF). The kinetics, memory, and reversibility of the switch were extensively characterized in Nicotiana benthamiana plants.© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

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.© 2017 Wiley Periodicals, Inc.

Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1463-1467, 2017.© 2017 American Institute of Chemical Engineers.

We have used the phiC31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than approximately 150 kb we have established a method that we call; 'Iterative Site Specific Integration' (ISSI). ISSI combines the activities of phiC31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid-HAC). We have demonstrated that the IIS-alphoid-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoid-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoid-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.

To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in-market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase-mediated cassette exchange (RMCE) system to build a site-specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT-flanked mAb expression cassette, we generated a clonal cell line with good productivity, long-term production stability, and low mAb gene-copy number indicating the vector was located in a 'hot-spot.' A SSI host cell line was made by removing the mAb genes from the 'hot-spot' by RMCE, creating a 'landing pad' containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP-based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened 'time-to-clinic' for therapeutic mAbs.© 2015 American Institute of Chemical Engineers.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).© 2021 American Institute of Chemical Engineers.

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 Wβ integrases, and show that Wβ 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 mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing.