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.

Regulatory mechanism of chicken lysozyme gene expression in oviducts examined using transgenic technology.

The use of promoters that strongly express target genes in the chicken oviduct is beneficial for the production of proteinaceous materials into egg white by transgenic chickens. To examine the regulatory mechanisms of chicken lysozyme gene expression in vivo, genetically manipulated chickens that express human erythropoietin under the control of a lysozyme promoter-enhancer were established. By using several deletion mutants of the promoter-flanking region, we found that a -1.9 kb DNase I hypersensitive site (DHS) was essential for oviduct-specific expression in genetically manipulated chickens. The concentration of human erythropoietin in egg white was 14-75 µg/ml, suggesting that the chicken lysozyme promoter containing -1.9 kb DHS is sufficient for the production of pharmaceuticals using transgenic chickens.

Primordial germ cell-specific expression of eGFP in transgenic chickens.

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked-in (gene-trapped) PGCs into the blood vessels of Hamburger-Hamilton stages (HH-stages) 13-16 chicken embryos. Gene-trapped chickens were established by crossing a chimeric chicken with a wild-type hen with very high efficiency. Heterozygous gene-trapped chickens grew normally and SSEA-1-positive cells expressed eGFP during HH-stages 13-30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene-trapped embryos obtained by crossing heterozygous gene-trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.

Siglec-F is induced by granulocyte-macrophage colony-stimulating factor and enhances interleukin-4-induced expression of arginase-1 in mouse macrophages.

Siglecs are cell surface lectins that recognize sialic acids and are primarily expressed in hematopoietic cells. Previous studies showed that some Siglecs regulate macrophage function. In the present study, we examined the induction and putative roles of mouse Siglec-F in bone-marrow-derived macrophages in mice. A quantitative RT-PCR analysis showed that the basal expression of Siglec-F was weak in bone-marrow-derived macrophages differentiated by macrophage colony-stimulating factor. However, a 24-hr stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced Siglec-F expression. GM-CSF also enhanced Siglec-F expression in thioglycollate-induced peritoneal macrophages. The inhibition of signal transducer and activator of transcription 5 (STAT5), but not that of phosphoinositide 3-kinase or mitogen-activated protein kinase kinase, significantly reduced the induction of Siglec-F. Interleukin-3, which uses a common ß-chain shared with the GM-CSF receptor to stimulate the STAT5 pathway, also enhanced Siglec-F expression. The knockdown of Siglec-F by a specific small interfering RNA enhanced GM-CSF-induced STAT5 phosphorylation, suggesting that Siglec-F down-regulates its own expression upon prolonged GM-CSF stimulation. Furthermore, the knockdown of Siglec-F reduced the STAT6 phosphorylation and expression of arginase-1 in interleukin-4-stimulated macrophages. These results suggest that Siglec-F fine-tunes the immune responses of macrophages.

PRDM14 and BLIMP1 control the development of chicken primordial germ cells.

The differentiation of primordial germ cells (PGCs) is a fundamental step in development. PR domain-containing protein 14 (PRDM14) and B lymphocyte-induced maturation protein 1 (BLIMP1) play pivotal roles in mouse PGC specification. In the present study, we assessed the roles of chicken orthologs of PRDM14 and BLIMP1 in PGC development. PRDM14 and BLIMP1 were expressed in blastodermal cells and PGCs. The in vivo knockdown of PRDM14 or BLIMP1 by introducing a replication-competent retroviral vector expressing shRNAs to the blastodermal stage of embryos reduced the number of SSEA-1 or chicken vasa homologue-positive PGCs on day 5.5-6.5. Since the inhibition of Activin receptor-like kinase 4/5/7 in cultured PGCs reduced the expression of PRDM14, BLIMP1, and NANOG, and that of MEK inhibited PRDM14 expression, the expression of these genes seems to be controlled by Activin A and FGF2 signaling. Overall, PRDM14, BLIMP1, and NANOG seem to be involved in the self-renewal of PGCs in cultured PGCs and embryos.

Molecular cloning of chicken TET family genes and role of chicken TET1 in erythropoiesis.

Ten-eleven translocation (TET) methylcytosine dioxygenase has potential as an active eraser to regulate the genomic DNA methylation status. We herein cloned chicken TET (cTET) family genes, and confirmed their functions. Quantitative reverse-transcription PCR showed that cTET1 was strongly expressed in erythrocytes throughout development. This cTET1 expression pattern, together with the results of methylated or hydroxymethylated DNA immunoprecipitation, suggests that cTET1 contributes to demethylation around the promoter region of the definitive-type ß-globin gene ßΑ in erythroid cells. The knockdown of cTET1 in T2ECs chicken erythroid progenitor cells suppressed the induction of ßΑ expression under differentiation conditions. These results suggest that cTET1 plays an important role in erythroid cell differentiation.

Characterization of chicken interferon-inducible transmembrane protein-10.

Interferon-inducible transmembrane protein (IFITM) family proteins are antivirus factors. In the present study, we examined the expression pattern of chicken IFITM10 using quantitative reverse transcription-polymerase chain reaction. In adult chickens, IFITM10 levels were markedly lower than those of IFITM3, which exhibits antivirus activity. On the other hand, IFITM10 was expressed in levels similar to those of IFITM3 in embryonic organs. Primordial germ cells in 2.5-d embryos expressed high levels of IFITM10, which gradually decreased with time. The interferon-α stimulation of embryonic fibroblast cells did not enhance the expression of IFITM10. The forced expression of IFITM10 slightly inhibited the infectivity of the VSV-G-pseudotyped lentiviral vector. Furthermore, cell fusion was inhibited by IFITM10 when HeLa cells transfected with the VSV-G expression vector were treated with low pH buffer. Although it remains unclear whether IFITM10 inhibits viral infections under physiological conditions, these results suggest that chicken IFITM10 exhibits antivirus activity.

Expression of interferon-inducible transmembrane proteins in the chicken and possible role in prevention of viral infections.

In mammals, interferon-inducible transmembrane proteins (IFITMs) prevent infections by various enveloped viruses. The expression of IFITMs in chicken was herein examined in the adult and embryonic organs using a quantitative reverse-transcription-polymerase chain reaction. The results obtained revealed that IFITM3 was expressed at a higher level than IFITM1, 2 and 5, in both embryonic and adult organs. However, the expression levels of IFITMs in embryonic organs were less than 5 % of those in adult lungs. Among the embryonic tissues examined, primordial germ cells (PGCs) at day 2.5 expressed relatively higher levels of IFITM3. IFITM3 expression levels were 1.5-fold higher in the chicken cell line DF-1 than in PGCs. The knockdown of IFITM3 in DF-1 cells by siRNA increased the infectivity of a vesicular stomatitis virus G protein-pseudotyped lentiviral vector, suggesting that lower levels of IFITM3 are still sufficient to restrict this viral vector.

Analyses of chicken sialyltransferases related to O-glycosylation.

The chicken ß-galactoside α2,3-sialyltransferase 1, 2, and 5 (ST3Gal1, 2, and 5) genes were cloned, and their enzymes were expressed in 293FT cells. ST3Gal1 and 2 exhibited enzymatic activities toward galactose-ß1,3-N-acetylgalactosamine and galactose-ß1,3-N-acetylglucosamine. ST3Gal5 only exhibited activity toward lactosylceramide. ST3Gal1 and 2 and previously cloned ST3Gal3 and 6 transferred CMP-sialic acid to asialofetuin. Reverse-transcription-quantitative PCR indicated that ST3Gal1 was expressed at higher levels in the trachea, lung, spleen, and magnum, and the strong expression of ST3Gal5 was observed in the spleen, magnum, and small and large intestines. ST3Gal1, 5, and 6 were also expressed in the tubular gland cells of the magnum, which secretes egg-white proteins. ST3Gal1, 5, and 6 were expressed in the egg chorioallantoic membrane, in which influenza viruses are propagated for the production of vaccines.

Analyses of chicken sialyltransferases related to N-glycosylation.

Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-ß1,4-N-acetylglucosamine (Galß1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.

Implication of SUMO E3 ligases in nucleotide excision repair.

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

Galactosylation of human erythropoietin produced by chimeric chickens expressing galactosyltransferase.

Human erythropoietin produced in the egg white of chimeric chicken contains N-glycan with lower amounts of terminal galactose and sialic acid; therefore, the chicken galactosyltransferase gene was introduced together with the human erythropoietin gene by a retroviral vector. We found that erythropoietin accumulated in the egg white was partially galactosylated.

SUMOylation of damaged DNA-binding protein DDB2.

Damaged DNA-binding protein (DDB) is a heterodimer composed of two subunits, p127 and p48, which have been designated DDB1 and DDB2, respectively. DDB2 recognizes and binds to UV-damaged DNA during nucleotide excision repair. Here, we demonstrated that DDB2 was SUMOylated in a UV-dependent manner, and its major SUMO E3 ligase was PIASy as determined by RNA interference-mediated knockdown. The UV-induced physical interaction between DDB2 and PIASy supported this notion. PIASy knockdown reduced the removal of cyclobutane pyrimidine dimers (CPDs) from total genomic DNA, but did not affect that of 6-4 pyrimidine pyrimidone photoproducts (6-4PPs). Thus, DDB2 plays an indispensable role in CPD repair, but not in 6-4PP repair, which is consistent with the observation that DDB2 was SUMOylated by PIASy. These results suggest that the SUMOylation of DDB2 facilitates CPD repair.

Moloney murine leukemia virus integrase and reverse transcriptase interact with PML proteins.

Pull-down assay and co-immunoprecipitation of cell extracts in which the integrase or reverse transcriptase of Moloney murine leukemia virus was transiently expressed showed that both enzymes interacted with PML proteins. In infected cells, interaction between the integrase and PML was also observed. Transient expression of PIASy and SUMO proteins facilitated SUMOylation of the integrase but had no apparent effects on the interaction with PML. A FLAG-tagged integrase co-localized with PML protein possibly in the PML body. Knockdown of PML by small interfering RNA resulted in reduced viral cDNA levels and integration efficiency. This suggested that PML proteins activated reverse transcription.

Constitutive expression of the brg1 gene requires GC-boxes near to the transcriptional start site.

We previously reported that BRG1, an ATPase subunit of SWI/SNF chromatin remodelling complexes, is constitutively expressed and that the alternative ATPase subunit (BRM) is inducibly expressed through differentiation in mammalian cells. In the present study, the regulatory elements that confer constitutive expression on brg1 were explored. First, we analysed the promoter proximal region surrounding its transcriptional start site. Using computer-aided analysis, a TATA-less, GC-rich promoter containing four putative binding sites for Sp1/3 was predicted. One of the putative Sp1/3-binding sites (from -21 to -15 bp) overlapped with a putative YY1-binding site. A gel-shift assay showed that YY1 but not Sp1/3 bound to this sequence and that Sp3 but not Sp1 bound to the other three predicted binding sites. Furthermore, chromatin immunoprecipitation analysis showed that Sp3 and YY1 bound to the promoter region together with TATA-binding protein in vivo. In vivo and in vitro binding assays showed that Sp3 and YY1 interacted with each other. Together, these results suggest that Sp3 and YY1 recruit general transcription factors and facilitate the assembly of a preinitiation complex.

Interactions between the nuclear matrix and an enhancer of the tryptophan oxygenase gene.

The gene for tryptophan oxygenase (TO) is expressed in adult hepatocytes in a tissue- and differentiation-specific manner. The TO promoter has two glucocorticoid-responsive elements (GREs), and its expression is regulated by glucocorticoid hormone in the liver. We found a novel GRE in close proximity to a scaffold/matrix attachment region (S/MAR) that was located around -8.5kb from the transcriptional start site of the TO gene by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays. A combination of nuclear fractionation and quantitative PCR analysis showed that the S/MAR was tethered to the nuclear matrix in both fetal and adult hepatocytes. ChIP assay showed that, in adult hepatocytes, the S/MAR-GRE and the promoter proximal regions interacted with lamin and heterogeneous nuclear ribonucleoprotein U in a dexamethasone dependent manner, but this was not the case in fetal cells, suggesting that developmental stage-specific expression of the TO gene might rely on the binding of the enhancer (the -8.5kb S/MAR-GRE) and the promoter to the inner nuclear matrix.

GATA4 inhibits expression of the tryptophan oxygenase gene by binding to the TATA box in fetal hepatocytes.

The glucocorticoid receptor regulates liver-specific expression of the tryptophan oxygenase gene through glucocorticoid responsive elements located -0.45 and -1.2 kb from the transcription start site. However, the hormone-mediated induction is restricted to adult hepatocytes, and fetal hepatocytes are unable to express the gene even in the presence of the receptor and glucocorticoid hormone. The difference in sensitivity to the hormone between adult and fetal hepatocytes has not been well understood. In this study, we analyzed the structure of the tryptophan oxygenase gene's promoter. The promoter has two TATA boxes, and transcription starts from the downstream TATA box. We found that a transcription factor GATA4 bound to the downstream TATA box and may inhibit the binding of TATA-binding protein, resulting in transcriptional repression even in the presence of glucocorticoid in fetal hepatocytes.

Sumoylation of CCAAT/enhancer-binding protein alpha and its functional roles in hepatocyte differentiation.

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Mammalian chromatin remodeling complex SWI/SNF is essential for enhanced expression of the albumin gene during liver development.

The chromatin remodeling complex SWI/SNF is known to regulate the transcription of several genes by controlling chromatin structure in an ATP-dependent manner. SWI/SNF contains the Swi2p/Snf2p like ATPases BRG1 or BRM exclusively. We found that the expression of BRM gradually increases and that of BRG1 decreases as liver cells differentiate. Chromatin immunoprecipitation assays revealed that the ATPase subunits of SWI/SNF and tumor suppressor retinoblastoma (RB) family proteins bind to the promoter region of the albumin gene in hepatocytes, and that the replacement of BRG1 with BRM and pRB with p130 at this site occurs over the course of differentiation. Small interfering RNA experiments showed that blocking the expression of BRG1 and BRM in fetal and adult hepatocytes, respectively, causes a reduction in albumin expression. In luciferase reporter assays with a pREP4-based reporter plasmid that forms a chromatin structure, BRG1 showed activity stimulating the expression of the albumin promoter mediated by CCAAT/enhancer-binding protein alpha (C/EBPalpha). This enhancement was facilitated by the RB family members pRB and p130. ATPase assays showed that both pRB and C/EBPalpha proteins directly stimulate the ATPase activity of BRG1. Our findings suggest that the mechanism by which the activity of transcription factors is enhanced by RB family members and SWI/SNF includes an increase in the ATPase activity of the chromatin remodeling complex.

YY1 binds to regulatory element of chicken lysozyme and ovalbumin promoters.

Chicken lysozyme is highly expressed in the oviduct. The 5' regulatory region of this gene contains a negative element that represses transcription. To assess the molecular basis underlying the regulation of lysozyme gene expression, we investigated the binding protein to this region. Sequence motif analysis suggested the existence of putative YY1 binding sites in this regulatory region. Electrophoretic mobility shift assay showed the specific binding of YY1 to the negative element. In addition, chromatin immunoprecipitation assay indicated that YY1 specifically bound to the negative element in oviduct cells but not in erythrocytes. It was suggested by electrophoretic mobility shift assay and chromatin immunoprecipitation assay that YY1 also bound to the negative regulatory region in the promoter of the ovalbumin gene which also shows oviduct-specific expression. Western blot analysis showed that YY1 was expressed in relatively high levels in the oviduct and nucleus fractionation experiments showed that YY1 was localized both in chromosome and nuclear matrix fractions. These results suggest that there are some specific roles in the negative regulatory regions of these genes in relation to the multifunctional transcription factor YY1.