Chicken Beta-actin Research Articles

Suitable Promoter for DNA Vaccination Using a pDNA Ternary Complex.

In this study, we evaluated the effect of several promoters on the transfection activity and immune-induction efficiency of a plasmid DNA (pDNA)/polyethylenimine/γ-polyglutamic acid complex (pDNA ternary complex). Model pDNAs encoding firefly luciferase (Luc) were constructed with several promoters, such as simian virus 40 (SV40), eukaryotic elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and chicken beta actin hybrid (CBh) (pSV40-Luc, pEF1-Luc, pCMV-Luc, and pCBh-Luc, respectively). Four types of pDNA ternary complexes, each with approximately 145-nm particle size and -30-mV ζ-potential, were stably constructed. The pDNA ternary complex containing pSV40-Luc showed low gene expression, but the other complexes containing pEF1-Luc, pCMV-Luc, and pCBh-Luc showed high gene expression in DC2.4 cells and spleen after intravenous administration. After immunization using various pDNA encoding ovalbumin (OVA) such as pEF1-OVA, pCMV-OVA, and pCBh-OVA, only the pDNA ternary complex containing pCBh-OVA showed significant anti-OVA immunoglobulin G (IgG) induction. In conclusion, our results showed that the CBh promoter is potentially suitable for use in pDNA ternary complex-based DNA vaccination.

Abstract PR001: Development and characterization of novel pre-clinical models of SFPQ-TFE3-driven renal cell carcinoma

Abstract Translocation Renal Cell Carcinomas (tRCC) are characterized by gene fusions that drive expression of chimeric TFE3 (Xp11.2) or TFEB (6p21.2). We developed and characterized a mouse model and inducible cell line expressing the SFPQ-TFE3 gene fusion to aid in biomarker and therapy development for this tumor type. We generated a murine knock-in model at the Rosa26 locus expressing the human SFPQ (exons 1-9) -TFE3 (exons 5-10) fusion gene downstream of a lox-stop-lox (LSL) sequence, under control of a chicken beta-actin promoter, on the C57BL/6 background. We crossed these LSL-SFPQ-TFE3 mice with the Cdh16-Cre model to induce SFPQ-TFE3 expression in renal epithelial cells. As an in vitro companion model, we generated HEK293 cells with stable doxycycline-inducible expression of the identical SFPQ-TFE3 fusion using the Flp-In T-RExTM system (SFPQ-TFE3-HEK293).Cdh16-Cre;LSL-SFPQ-TFE3 mice had reduced survival compared to the wild-type mice (p<0.0001) and none survived past P19. At P1, Cdh16-Cre;LSL-SFPQ-TFE3 mice had scattered mild tubular dilation, with strong nuclear TFE3 protein expression in distal tubules and collecting ducts that was absent in controls. By P15, Cdh16-Cre;LSL-SFPQ-TFE3 kidneys were 30% larger than wild-type mice (p<0.0001), with diffuse and strong nuclear expression of TFE3 throughout the majority of renal tubules. At this timepoint, Cdh16-Cre;LSL-SFPQ-TFE3 kidneys had prominent intratubular proliferation of renal epithelial cells, occasionally occluding the lumen, with diffuse psammomatous intraluminal calcification and mild patchy tubular dilatation. Glomerular morphology and size were not significantly different between the two groups at P15. However, the Cdh16-Cre;LSL-SFPQ-TFE3 mice had a lower density of glomeruli compared to the wild-type controls (5 glomeruli/mm² vs 50 glomeruli/mm²; p=0.03), potentially reflecting tubular expansion or aberrant glomerulogenesis. Wild-type and Cdh16-Cre;LSL-SFPQ-TFE3 mice exhibited similar tubular proliferative rate at P1 as assessed by Ki67. By P7, Cdh16-Cre;LSL-SFPQ-TFE3 mice showed significantly higher expression of Ki67 (p=0.03) but by P15, Ki67 index was lower (p=0.004) in the Cdh16-Cre;LSL-SFPQ-TFE3 mice compared to controls. Interestingly, by 48 hours after doxycycline induction, SFPQ-TFE3-HEK293 cells showed significantly decreased proliferation by MTT assay compared to the same cells without doxycycline treatment or to the parental Flp-In line treated with doxycycline (p=0.0002). A novel transgenic model for SFPQ-TFE3-driven tRCC shows early postnatal lethality, likely due to renal failure, precluding aging of these mice for tumor development. Though tubular proliferation is initially increased with SFPQ-TFE3 expression, Ki-67 expression is reduced by P15, paralleling the reduction in growth seen in inducible SFPQ-TFE3-HEK293 cells. These data suggest that SFPQ-TFE3 expression may paradoxically result in oncogene-induced senescence or cell cycle arrest in renal cells and further studies are underway to test this hypothesis. Citation Format: Adrianna Amaral de Aragao Mendes, Hans Liu, Juhyung Woo, Kaushal Asrani, Avi Rosenberg, Pedram Argani, Tamara Lotan. Development and characterization of novel pre-clinical models of SFPQ-TFE3-driven renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR001.

Subcutaneously Transplanted Fresh Cartilage in Allogeneic and Xenogeneic Immunocompetent Mouse.

Cartilage is considered to be immune privileged in general. Clinically, live cells are removed from subcutaneously transplanted allogeneic cartilage mainly for preservation and for infection control. However, because maintaining cartilage feature requires live chondrocyte, it would be beneficial to subcutaneously transplant cartilage with live chondrocyte even if it was allogeneic. We harvested femoral head from 3-week-old male C57BL/6 mice, subcutaneously transplanted to 6-week-old male mice, BALB/c, BALB/c nu/nu, or C57BL/6-Tg (enhanced green fluorescent protein [EGFP] under the control of the CMV-IE enhancer, chicken beta-actin promoter, rabbit beta-globin genomic DNA [CAG promoter]), as allogeneic, allogeneic immunodeficient control, or syngeneic transplantation. We also transplanted cartilaginous particles from human induced pluripotent stem cells derived from human leukocyte antigen homozygous donor to 6-week-old male mice either BALB/c and BALB/c nu/nu as xenogeneic or xenogeneic immunodeficient control. The transplantation periods were 1, 2, 3, 4, 8, 12, and 24 weeks. As the result, we did not observe exposure of the transplant or apparent macroscopic inflammatory in all samples. Histological analysis suggested that the femoral head showed focal ossification and thinning in syngeneic transplantation. In allogeneic transplantation, slight invasion of CD3 (+) T cell and the denaturation of the cartilage were observed, suggesting immune reaction against allogeneic cartilage. In xenogeneic transplantation, slight invasion of CD3 (+) cell and CD4 (+) cell and the structure of the perichondrium-like tissue got unclear, suggesting slight immune reaction against xenogeneic cartilage. Our findings suggest that we should carefully investigate for appropriate procedure to control immune reaction against allogeneic cartilage with live chondrocyte and to maintain its cartilage feature for long time.

A systematic comparison of optogenetic approaches to visual restoration

During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation.

The CAG promoter maintains high-level transgene expression in HEK293 cells.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human-like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT-PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33°C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.

JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

Adeno-Associated Virus Capsid-Promoter Interactions in the Brain Translate from Rat to the Nonhuman Primate.

Recently, we established an adeno-associated virus (AAV9) capsid-promoter interaction that directly determined cell-specific gene expression across two synthetic promoters, Cbh and CBA, in the rat striatum. These studies not only expand this capsid-promoter interaction to include another promoter in the rat striatum but also establish AAV capsid-promoter interactions in the nonhuman primate brain. When AAV serotype 9 (AAV9) vectors were injected into the rat striatum, the minimal synthetic promoter JetI drove green fluorescent protein (GFP) gene expression predominantly in oligodendrocytes. However, similar to our previous findings, the insertion of six alanines into VP1/VP2 of the AAV9 capsid (AAV9AU) significantly shifted JetI-driven GFP gene expression to neurons. In addition, previous retrograde tracing studies in the nonhuman primate brain also revealed the existence of a capsid-promoter interaction. When rAAV2-Retro vectors were infused into the frontal eye field (FEF) of rhesus macaques, local gene expression was prominent using either the hybrid chicken beta actin (CAG) or human synapsin (hSyn) promoters. However, only the CAG promoter, not the hSyn promoter, led to gene expression in the ipsilateral claustrum and contralateral FEF. Conversely, infusion of rAAV2-retro-hSyn vectors, but not rAAV2-retro-CAG, into the macaque superior colliculus led to differential and selective retrograde gene expression in cerebellotectal afferent cells. Clearly, this differential promoter/capsid expression profile could not be attributed to promoter inactivation from retrograde transport of the rAAV2-Retro vector. In summary, we document the potential for AAV capsid/promoter interactions to impact cell-specific gene expression across species, experimental manipulations, and engineered capsids, independent of capsid permissivity.

Mosaic Recombinant Adeno-associated Virus Vector rAAV/DJ/CAG for Targeted Gene Delivery to Melanoma Cells Metastasized to the Lung

Patients with metastasized melanoma have limited treatment options and poor diagnosis. Therefore, the development of treatments requires a new therapeutic approach, of which gene therapy using rAAV vectors can be proposed. The aim of the study was to examine the efficiency of the rAAV vector to transduce mouse melanoma cells both in vitro and in vivo. Different rAAV serotypes encoding GFP under the control of both chicken beta-actin and cytomegalovirus promoters were used in the experiments. Intranasal, intraperitoneal, intravenous and intratumoral pathways of administration of rAAV vectors were tested using quantitative-PCR and immunohistochemical staining. The highest transduction efficiency in metastatic cells in vivo was observed 7 days after intranasal administration of a 1010 gc/0.03 ml dose of rAAV/DJ-CAG. Melanoma gene therapy based on rAAV vectors is a possible treatment option.

High-Level Expression of Alkaline Phosphatase by Adeno-Associated Virus Vector Ameliorates Pathological Bone Structure in a Hypophosphatasia Mouse Model

Hypophosphatasia (HPP) is a systemic skeletal disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). We recently reported that survival of HPP model mice can be prolonged using an adeno-associated virus (AAV) vector expressing bone-targeted TNALP with deca-aspartate at the C terminus (TNALP-D10); however, abnormal bone structure and hypomineralization remained in the treated mice. Here, to develop a more effective and clinically applicable approach, we assessed whether transfection with TNALP-D10 expressing virus vector at a higher dose than previously used would ameliorate bone structure defects. We constructed a self-complementary AAV8 vector expressing TNALP driven by the chicken beta-actin (CBA) promoter (scAAV8-CB-TNALP-D10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 1012 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 1012 v.g./body-treated HPP mice grew normally and displayed improved bone structure at the knee joints in X-ray images. Micro-CT analysis showed normal trabecular bone structure and mineralization. The mechanical properties of the femur were also recovered. Histological analysis of the femurs demonstrated that ALP replacement levels were sufficient to promote normal, growth plate cartilage arrangement. These results suggest that AAV vector-mediated high-dose TNALP-D10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP.

Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

Transduction of Adeno-Associated Virus Vectors Targeting Hair Cells and Supporting Cells in the Neonatal Mouse Cochlea.

Adeno-associated virus (AAV) is the preferred vector for gene therapy of hereditary deafness, and different viral serotypes, promoters and transduction pathways can influence the targeting of AAV to different types of cells and the expression levels of numerous exogenous genes. To determine the transduction and expression patterns of AAV with different serotypes or promoters in hair cells and supporting cells in the neonatal mouse cochlea, we examined the expression of enhanced green fluorescent protein (eGFP) for five different types of AAV vectors [serotypes 2, 9, and Anc80L65 with promoter cytomegalovirus (CMV)-beta-Globin and serotypes 2 and 9 with promoter chicken beta-actin (CBA)] in in vitro cochlear explant cultures and we tested the transduction of AAV2/2-CBA, AAV2/9-CBA, and AAV2/Anc80L65-CMV by in vivo microinjection into the scala media of the cochlea. We found that each AAV vector had its own transduction and expression characteristics in hair cells and supporting cells in different regions of the cochlea. There was a tonotopic gradient for the in vitro transduction of AAV2/2-CBA, AAV2/9-CBA, AAV2/2-CMV, and AAV2/9-CMV in outer hair cells (OHCs), with more OHCs expressing eGFP at the base of the cochlea than at the apex. AAV2/2-CBA in vitro and AAV2/Anc80L65-CMV in vivo induced more supporting cells expressing eGFP at the apex than in the base. We found that AAV vectors with different promoters had different expression efficacies in hair cells and supporting cells of the auditory epithelium. The CMV-beta-Globin promoter could drive the expression of the delivered construct more efficiently in hair cells, while the CBA promoter was more efficient in supporting cells. The in vitro and in vivo experiments both demonstrated that AAV2/Anc80L65-CMV was a very promising vector for gene therapy of deafness because of its high transduction rates in hair cells. These results might be useful for selecting the appropriate vectors for gene delivery into different types of inner ear cells and thus improving the effectiveness of gene therapy.

Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1-9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons.

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method

BackgroundRecently, we demonstrated the three-dimensional (3D) localization of murine trophoblast giant cells in the pregnant uterus using a modified Clear Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) tissue-clearing method and hybrid construct consisting of the cytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG) conjugated enhanced green fluorescent protein (EGFP) transgenic mice. In this study, we applied this method to obtain a transparent whole-image of the ovary and observed the 3D localization of individual oocytes in the developing follicles.MethodsOvarian samples were obtained from EGFP transgenic mice and subjected to nuclear staining with propidium iodide (PI) and CUBIC treatment. The detection of double fluorescence signals (green and red) and subsequent reconstruction of 3D images of the whole ovary were performed by light-sheet microscopy and computer programs, respectively.ResultsThe ovary became transparent using the CUBIC method and each nucleus of the follicle component cells was uniformly fluoro-stained by PI perfusion. In contrast, EGFP signals were strong in oocytes, whereas those of surrounding granulosa cells were faint. These signal differences in EGFP expression among oocytes, granulosa cells, and theca-interstitial cells produce well-contrasted images of the growing follicles, providing clear information of the 3D localization of individual oocytes.ConclusionThese results indicate that this procedure is one of the effective approaches to analyze the 3D structure of follicles in the whole ovary.

Imaging of electrical activity in small diameter fibers of the murine peripheral nerve with virally-delivered GCaMP6f

Current neural interfaces are hampered by lack of specificity and selectivity for neural interrogation. A method that might improve these interfaces is an optical peripheral nerve interface which communicates with individual axons via optogenetic reporters. To determine the feasibility of such an interface, we delivered the genetically encoded calcium indicator GCaMP6f to the mouse peripheral nerve by intramuscular injection of adenoassociated viral vector (AAV1) under the control of the CAG (chicken beta actin- cytomegalovirus hybrid promoter). Small diameter axons in the common peroneal nerve were transduced and demonstrated electrically inducible calcium transients ex vivo. Responses to single electrical stimuli were resolvable, and increasing the number of stimuli resulted in a monotonic increase in maximum fluorescence and a prolongation of calcium transient kinetics. This work demonstrates the viability of using a virally-delivered, genetically-encoded calcium indicator to read-out from peripheral nerve axons.

Characterisation of a stably integrated expression system for exogenous protein expression in DT40

The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV, CBA or CAG, and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful experimental tool for future DT40 experiments.

P.355 - Mitochondrial dysfunction in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by ptosis, dysphagia and weakness of proximal limbs. OPMD is caused by the expansion of polyalanine in poly(A)-binding protein, nuclear 1 (PABPN1). The molecular pathogenesis is still poorly understood. As for today, mitochondrial abnormality has been proposed as the possible etiology. The aim of our study is to evaluate mitochondrial function in animal and cell models of OPMD, and to realize the involvement of mitochondrial dysfunction in the pathogenesis of OPMD. First, we employed transgenic mice expressing high level of expanded human PABPN1 with 13-alanine stretch driven by cytomegalovirus early enhancer element/ chicken beta actin (CAG) promoter. Immunofluorescence staining of skeletal muscles showed that PABPN1 co-localized with mitochondria. PABPN1 was detected in mitochondria fraction from the skeletal muscle, and PABPN1 located at the cytosolic face of outer membrane of mitochondria. Expression levels and enzymatic activities of respiratory chain complex proteins were reduced in the skeletal muscle of transgenic mice, compared with non-transgenic littermates. Next, we used cell model expressing PABPN1 with 10-alanine and 18-alanine stretch. We also observed mitochondrial dysfunction in cells with expanded polyalanine stretch. We proposed that expansion of polyalanine in PABPN1 may cause mitochondrial dysfunction, and it may be a possible mechanism of myodegeneration in OPMD.

RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Abstract 185: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 in Mice Lowers Asymmetric Dimethylarginine and Improves Vasomotor Function

Background: It has been demonstrated in various studies that ADMA (asymmetric dimethylarginine), an inhibitor of nitric oxide synthase, is associated with the increased risk of cardiovascular diseases. There are two known pathways of ADMA metabolism: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolases (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The second pathway is still poorly understood. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and improvement of vasomotor function. Methods and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. qPCR and Western Blot were used to confirm the ubiquitous expression of the transgene. HPLC-MS/MS was used to generate biochemical data. Systemic ADMA levels were decreased by 15% (p&lt;0.05) in the TG mice, whereas ADGV plasma levels were six times higher in comparison with wild type animals (p&lt;0.001). Heart and lung of TG animals exhibited 2 times lower tissue ADMA content in comparison with wild type littermates (p&lt;0.05). In further experiments, we crossed the AGXT2 TG mice with DDAH1 KO mice and showed that upregulation of AGXT2 protects DDAH1 KO mice from elevation of plasma ADMA levels and restores endothelium-dependent vasodilation in aortic rings. In the current experiments we are assessing whether AGXT2 overexpression also protects DDAH1 KO mice from hypertension. Conclusion: In the current study we demonstrated that upregulation of AGXT2 leads to lowering of ADMA levels and improvement of endothelium-dependent relaxation in vivo in the settings of DDAH1 deficiency . AGXT2 thereby may be a potential drug target for long-term reduction of systemic ADMA levels in cardiovascular pathologies. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far.

Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies.

Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model.