Design and validation of functionalized redox-responsive hydrogel beads for high-throughput screening of antibody-secreting mammalian cells.

Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

Inducible transgene expression in CHO cells using an artificial transcriptional activator with estrogen-binding domain.

Biopharmaceuticals, including therapeutic antibodies, are rapidly growing products in the pharmaceutical market. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are widely used as production hosts because recombinant antibodies require complex three-dimensional structures modified with sugar chains. Recombinant protein production using mammalian cells is generally performed with cell growth. In this study, we developed a technology that controls cell growth and recombinant protein production to induce recombinant protein production with predetermined timing. Expression of green fluorescent protein (GFP) gene and a single-chain antibody fused with the Fc-region of the human IgG1 (scFv-Fc) gene can be induced and mediated by the estrogen receptor-based artificial transcription factor Gal4-ERT2-VP16 and corresponding inducer drugs. We generated CHO cells using an artificial gene expression system. The addition of various concentrations of inducer drugs to the culture medium allowed control of proliferation and transgene expression of the engineered CHO cells. Use of 4-hydroxytamoxifen, an antagonist of estrogen, as an inducing agent yielded high gene expression at a concentration more than 10-fold lower than that of ß-estradiol. When scFv-Fc was produced under inducing conditions, continuous production was possible for more than 2 weeks while maintaining high specific productivity (57 pg cell-1 day-1 ). This artificial gene expression control system that utilizes the estrogen response of estrogen receptors can be an effective method for inducible production of biopharmaceuticals.

High-Level Production of scFv-Fc Antibody Using an Artificial Promoter System with Transcriptional Positive Feedback Loop of Transactivator in CHO Cells.

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems.

Emerging roles for diguanylate cyclase during the evolution of soma in dictyostelia.

BACKGROUND: Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4. RESULTS: Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells. CONCLUSIONS: The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.

Development of the dictyostelid Polysphondylium violaceum does not require secreted cAMP.

Group 4 Dictyostelia, like Dictyostelium discoideum, self-organize into aggregates and fruiting bodies using propagating waves of the chemoattractant cAMP, which are produced by a network containing the adenylate cyclase AcaA, cAMP receptors (Cars) and the extracellular cAMP phosphodiesterase PdsA. Additionally, AcaA and the adenylate cyclases AcrA and AcgA produce secreted cAMP for induction of aggregative and prespore gene expression and intracellular cAMP for PKA activation, with PKA triggering initiation of development and spore and stalk maturation. Non-group 4 species also use secreted cAMP to coordinate post-aggregative morphogenesis and prespore induction but use other attractants to aggregate. To understand how cAMP's role in aggregation evolved, we deleted the acaA, carA and pdsA genes of Polysphondylium violaceum, a sister species to group 4. acaA- fruiting bodies had thinner stalks but otherwise developed normally. Deletion of acrA, which was similarly expressed as acaA, reduced aggregation centre initiation and, as also occurred after D. discoideum acrA deletion, caused spore instability. Double acaA-acrA- mutants failed to form stable aggregates, a defect that was overcome by exposure to the PKA agonist 8Br-cAMP, and therefore likely due to reduced intracellular cAMP. The carA- and pdsA- mutants showed normal aggregation and fruiting body development. Together, the data showed that P. violaceum development does not critically require secreted cAMP, while roles of intracellular cAMP in initiation of development and spore maturation are conserved. Apparently, cell-cell communication underwent major taxon-group specific innovation in Dictyostelia.

Adenylate cyclase A amplification and functional diversification during Polyspondylium pallidum development.

BACKGROUND: In Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling. RESULTS: In contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2'H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1- cells showed relatively thin stalks, aca2- showed delayed secondary sorogen formation and aca3- formed less aggregation centers. The aca1-aca2- and aca1-aca3- mutants combined individual defects, while aca2-aca3- and aca1-aca3-aca2- additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2-aca3- and aca1-aca3-aca2- by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis aca-/ACG cells produced a pulse of cAMP upon stimulation with 2'H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone. CONCLUSIONS: The three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1-aca3-aca2- mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour.

Promotion of Cyst Formation from a Renal Stem Cell Line Using Organ-Specific Extracellular Matrix Gel Format Culture System.

Researchers have long awaited the technology to develop an in vitro kidney model. Here, we establish a rapid fabricating technique for kidney-like tissues (cysts) using a combination of an organ-derived extracellular matrix (ECM) gel format culture system and a renal stem cell line (CHK-Q cells). CHK-Q cells, which are spontaneously immortalized from the renal stem cells of the Chinese hamster, formed renal cyst-like structures in a type-I collagen gel sandwich culture on day 1 of culture. The cysts fused together and expanded while maintaining three-dimensional structures. The expression of genes related to kidney development and maturation was increased compared with that in a traditional monolayer. Under the kidney-derived ECM (K-ECM) gel format culture system, cyst formation and maturation were induced rapidly. Gene expressions involved in cell polarities, especially for important material transporters (typical markers Slc5a1 and Kcnj1), were restored. K-ECM composition was an important trigger for CHK-Q cells to promote kidney-like tissue formation and maturation. We have established a renal cyst model which rapidly expressed mature kidney features via the combination of K-ECM gel format culture system and CHK-Q cells.

Recent advances in animal cell technologies for industrial and medical applications.

The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.

HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions.

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems.

Novel cell lines derived from Chinese hamster kidney tissue.

Immortalized kidney cell lines are widely used in basic and applied research such as cell permeability tests and drug screening. Although many cell lines have been established from kidney tissues, the immortalization process has not been clarified in these cell lines. In this study, we analyzed the phenotypic changes that occurred during the immortalization of kidney cells derived from Chinese hamster tissue in terms of karyotype and gene expression profiles. In the newly established cell line, designated as CHK-Q, gene expression profiles at each stage of the immortalization process and during the adaptation to serum-free conditions were analyzed by DNA microarray. Renal stem cell markers CD24 and CD133 were expressed in CHK-Q cells, suggesting that CHK-Q cells were transformed from renal stem cells. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to identify the pathways of upregulated and downregulated genes revealed that the immortalization of CHK-Q cells was associated with increased fluctuations in the expression of specific proto-oncogenes. Karyotype analysis of spontaneously immortalized CHK-Q cells indicated that CHK-Q chromosomes had a typical modal number of 23 but possessed slight chromosomal abnormalities. In this study, we investigated the mechanism of cell environmental adaptation by analyzing gene expression behavior during the immortalization process and serum-free adaptation. CHK-Q cells are applicable to the fields of biotechnology and biomedical science by utilizing their characteristics as kidney-derived cells.

Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.

Hypoxia-responsive expression of vascular endothelial growth factor for induction of angiogenesis in artificial three-dimensional tissues.

Constructing three-dimensional (3D) tissues is an important process to improve cellular functions in tissue engineering. When transplanting artificially constructed tissues, a poor vascular network restricts oxygen and nutrient supplies to the tissue cells, which leads to cell death and reduced rates of tissue engraftment. Therefore, it is necessary to develop a system that builds a vascular network within 3D tissues. Here, we developed a hypoxia-responsive gene expression system for production of an angiogenic factor, vascular endothelial growth factor (VEGF), to improve hypoxia and nutrition deficiencies inside artificial 3D tissues. We demonstrated that cells into which the hypoxia-responsive VEGF gene expression system had been introduced autonomously controlled VEGF expression in a hypoxic stress-dependent manner. Next, we confirmed that VEGF expression within a 3D cell sheet was induced in response to a hypoxic environment in vitro. The genetically modified cell sheet was subcutaneously transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results suggest that the hypoxia-responsive VEGF gene expression system is promising to prepare artificial 3D tissues in regenerative medicine.

Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system.

The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

Hepatoma cells are a promising cell source for the construction of bioartificial liver (BAL) systems owing to their high proliferative capability. However, their low liver function compared with primary hepatocytes is a major problem. In a previous study, we established a genetically modified hepatoma cell line, Hepa/8F5, in which eight liver-enriched transcription factor (LETF) genes were transduced into mouse hepatoma Hepa1-6 cells using a drug-inducible transactivator system. These cells proliferate actively under normal culture conditions, meaning that large quantities can be prepared easily. When the overexpression of the LETFs is induced by the addition of an inducer drug, cell growth stops and cell morphology changes with concomitant high expression of liver functions. However, the liver functions largely depend on the presence of the inducer drug, which must be continuously added to maintain these enhanced functions. In the present study, we attempted to modify the method of induction of LETF overexpression in Hepa/8F5 cells to remove the requirement for continual drug addition. To this end, we constructed a system in which the artificial transactivator was transcribed and amplified under the control of a heat-shock protein promoter, and introduced the system into the genome of Hepa/8F5 cells. In our modified cell line, heat-triggered LETF expression was confirmed to induce high liver function. After drug-screening of transfected cells, we established a hepatoma cell line (Hepa/HS), which exhibited high, heat-inducible liver functions. The Hepa/HS cells may represent a new cell source for hepatic studies such as the construction of BAL systems. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells.

Retrotransposons, such as long interspersed element-1 (LINE-1), can copy themselves to other genomic loci via a transposition event (termed retrotransposition). Retrotransposons, therefore, have potential use as an efficient gene delivery tool to integrate multiple copies of a target gene into a host genome. Here, we developed a retrotransposon vector based on LINE-1 that achieves target gene integration of multiple transgene copies. The retrotransposon vector contains a neomycin resistance gene split by an intron as a marker gene, and a gene encoding an antibody single-chain variable fragment (Fv) fused with the constant antibody region (Fc) (scFv-Fc) as a model target gene. G418-resistant Chinese hamster ovary cells were generated using this retrotransposon vector, and scFv-Fc was produced in the culture medium. To regulate retrotransposition, we developed a retrotransposon vector system that separately expressed the two open reading frames (ORF1 and ORF2) of LINE-1. Genomic PCR analysis detected the transgene sequence in almost all tested clones. Compared with clones established using the intact LINE-1 vector, clones generated with the split ORF1 and ORF2 system showed similar specific scFv-Fc productivity and retrotransposition efficiency. This approach of using a retrotransposon-based vector system has the potential to provide a new gene delivery tool for mammalian cells.

Miniaturized skeletal muscle tissue fabrication for measuring contractile activity.

The contractile function of skeletal muscle is essential for maintaining the vital activity of life. Muscular diseases such as muscular dystrophy severely compromise the quality of life of patients and ultimately lead to death. There is therefore an urgent need to develop therapeutic agents for these diseases. In a previous study, we showed that three-dimensional skeletal muscle tissues fabricated using the magnetic force-based tissue engineering technique exhibited contractile activity, and that drug effects could be evaluated based on the contractile activity of the skeletal muscle tissues. However, the reported method requires a large number of cells and the tissue preparation procedure is complex. It is therefore necessary to improve the tissue preparation method. In this study, a miniature device made of polydimethylsiloxane was used to simplify the production of contracting skeletal muscle tissues applicable to high-throughput screening. The effects of model drugs on the contractile force generation of skeletal muscle tissues prepared from mouse C2C12 myoblast and human induced pluripotent stem cells were evaluated using the miniature muscle device. The results indicated that the muscle device system could provide a useful tool for drug screening.

Loss of the Polyketide Synthase StlB Results in Stalk Cell Overproduction in Polysphondylium violaceum.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.

Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration.

It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.