Puromycin Resistance Gene Research Articles

Decreasing immunogenicity of ESC derived cardiomyocytes (73.1)

Abstract One of the major obstacles to repair damaged myocardium with embryonic stem cell derived cardiomyocytes (ESC-CM) is their rejection by non-syngeneic hosts. We aimed to reduce the immunogenicity of these cells by downregulation of MHC class I molecules (MHC-I) on their surface. Cardiomyocytes were derived from mouse ESC in which puromycin resistance gene is driven by a cardiac-specific promoter. They formed beating cardiomyocyte layers and expressed cardiac specific proteins. Interferon gamma treatment was used to mimic the effects of inflammatory stimuli on the expression of MHC-I. Transduction of ESC-CM with beta-2-microglobulin shRNA decreased expression of MHC-I in both interferon gamma-treated and untreated samples. When MHC-I downregulated ESC-CM were used as in vitro targets, activation of cognate T-lymphocytes was significantly reduced. Moreover, in vivo, MHC-I downregulated cells exhibited improved survival when injected i.p. into allogeneic, but not to syngeneic mice. To confirm that this strategy can be used for different cell types, the results were verified using W19 fibroblasts. In the past, ESC-CM have been shown to improve ischemic myocardium in syngeneic or immunocompromised animals. Inhibition of MHC-I via shRNA offers a simple approach to minimize the immunogenicity of these cells for use in allo- or xenogeneic recipients. Extension of this methodology to ESC of larger mammals can provide a source of less immunogenic cardiomyocytes for human heart repair.

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies

Genomic libraries constructed in the original Bacterial Artificial Chromosome (BAC) and P1 Artificial Chromosome (PAC) cloning systems were very useful for completion of the Human Genome Project. Libraries constructed in these vector systems were used to generate physical maps of all twenty three-chromosome pairs and served as the templates for DNA sequencing. The PAC vector pJCPAC-Mam2 is a 23 kilobase (kb) shuttle vector that is highly versatile and useful for functional studies in human tissue culture cell lines. pJCPAC-Mam2 contains the P1 single-copy replicon for low copy expression and a multi-copy lytic replicon under the control of the lac repressor for high copy expression in Escherichia coli, wild type and mutant loxP sites for generation of bidirectional nested deletions in any clone of interest and the Epstein Barr Virus (EBV) latent replication origin oriP, the Epstein Barr Nuclear Antigen 1 (EBNA1) gene, and a puromycin-resistance gene for propagation in mammalian cells. We constructed an arrayed 115,000-member human genomic library in pJCPAC-Mam2 that is housed in twelve hundred 96 well dishes. Subsequent pooling using a column and rows strategy reduced the library to 19 final pools for Polymerase Chain Reaction (PCR) screening. We estimate that the average insert size is approximately 57 kb based on NotI digestion and Field Inversion Gel Electrophoresis (FIGE) analysis of 473 random PAC clones. The library appears to represent a two- to three-fold coverage of the human genome based on screening of the library with PCR primers representing nine genes. This library should serve as a valuable resource to validate potential disease genes identified by genome-wide association studies (GWAS) in human tissue culture cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.

Generation of Mouse STO Feeder Cell Lines That Confer Resistance to Several Types of Selective Drugs.

Feeder cells are generally required for establishment and maintenance of embryonic stem (ES)/induced pluripotent stem (iPS) cells. Increased demands for generation of those cells carrying various types of vectors (i.e., KO vectors and transgenes) also require feeder cells that confer resistance to any types of preexisting selective drugs. Unfortunately, the use of the feeders that are resistant to various drugs appears to be limited to a few laboratories. Here we generated a set of gene-engineered STO feeder cells that confer resistance to several commercially available drugs. The STO cells, which have long been used as a feeder for mouse ES and embryonal carcinoma (EC) cells, were transfected with pcBIH [carrying bleomycin resistance gene (ble) and hygromycin B phosphotransferase gene (Hyg)], pcBIP [carrying ble and puromycin resistance gene (puro)], or pcBSN [carrying ble and neomycin resistance gene (neo)]. The resulting stably transfectants (termed SHB for pcBIH, SPB for pcBIP, and SNB for pcBSN) exhibited bleomycin/hygromycin, bleomycin/puromycin, or bleomycin/neomycin, as expected. The morphology of these cells passaged over 18 generations was indistinguishable from that of parental STO cells. Of isolated clones, the SHB3, SPB3, and SNB2 clones successfully supported the growth of mouse ES cells in an undifferentiated state, when coculture was performed. PCR analysis revealed the presence of the selective markers in these clones, as expected. These SHB3, SPB3, and SNB2 cells will thus be useful for the acquisition and maintenance of genetically manipulated ES/iPS cells.

Transposon mediated co-integration and co-expression of transgenes in CHO-DG44 cells

Background Transposon systems mediate stable integration of exogenous DNA elements into a host cell genome, and have been successfully used in mammalian cells for the generation of stable cell lines. The piggyBac (PB) transposon system has been shown to have several advantages over the other transposon system available [1-3]. It has also been shown to generate stable cell lines at significantly higher frequency than the conventional transfections [3]. Here, we investigated the efficiency of the piggyBac (PB) transposon to facilitate the co-expression of multiple artificial transposons, each bearing a single transgene and the puromycin resistance gene for selection. Green fluorescent protein (eGFP), red fluorescent protein (mKate), and a human IgG1 antibody were used as model proteins [4]. The effect of the stringency of selection on pool productivity was determined with increasing concentrations of puromycin. The duration of selection necessary for the generation of recombinant cell pools was also tested by selecting for a period of either 5 or 10 days.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) Protein Regulates Osteoblast Differentiation

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is an established regulator of tissue mineralization. Previous studies demonstrated that ENPP1 is expressed in differentiated osteoblasts and that ENPP1 influences matrix mineralization by increasing extracellular levels of inorganic pyrophosphate. ENPP1 is also expressed in osteoblastic precursor cells when stimulated with FGF2, but the role of ENPP1 in preosteoblastic and other precursor cells is unknown. Here we investigate the function of ENPP1 in preosteoblasts. We find that ENPP1 expression is critical for osteoblastic differentiation and that this effect is not mediated by changes in extracellular concentration levels of phosphate or pyrophosphate or ENPP1 catalytic activity. MC3T3E1(C4) preosteoblastic cells, in which ENPP1 expression was suppressed by ENPP1-specific shRNA, and calvarial cells isolated from Enpp1 knock-out mice show defective osteoblastic differentiation upon stimulation with ascorbate, as indicated by a lack of cellular morphological change, a lack of osteoblast marker gene expression, and an inability to mineralize matrix. Additionally, MC3T3E1(C4) cells, in which wild type or catalytic inactive ENPP1 expression was increased, exhibited an increased tendency to differentiate, as evidenced by increased osteoblast marker gene expression and increased mineralization. Notably, treatment of cells with inorganic phosphate or pyrophosphate inhibited, as opposed to enhanced, expression of multiple genes that are expressed in association with osteoblast differentiation, matrix deposition, and mineralization. Our results indicate that ENPP1 plays multiple and distinct roles in the development of mineralized tissues and that the influence of ENPP1 on osteoblast differentiation and gene expression may include a mechanism that is independent of its catalytic activity.

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes (1-3). The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression(4,5). These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues (6). The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis (7-10). CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance (11). We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

The Third Transmembrane Segment of Orai1 Protein Modulates Ca2+ Release-activated Ca2+ (CRAC) Channel Gating and Permeation Properties

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Self-renewal gene tracking to identify tumour-initiating cells associated with metastatic potential

Tumour-initiating cells (TICs) are rare cancer cells isolated from tumours of different origins including high-grade tumours that sustain neoplasic progression and development of metastatic disease. They harbour deregulated stem cells pathways and exhibit an unchecked ability to self-renew, a property essential for tumour progression. Among the essential factors maintaining embryonic stem (ES) cells properties, OCT-4 (also known as POU5F1) has been detected in tumours of different origins. Although ectopic expression results in dysplasic growth restricted to epithelial tissues, overexpression expands the proportion of immature cells in teratomas. However, OCT-4-expressing cells have not been purified from spontaneously occurring tumours, thus information concerning their properties is rather scant. Here, using p53-/- mice expressing green fluorescent protein and the puromycin resistance gene under the control of the Oct-4 promoter, we show that OCT-4 is expressed in 5% onwards of the undifferentiated tumour cell populations derived from different organs. OCT-4 expression was low as compared with ES cells, but was associated with a 'stemness' signature and expression of the chemokine receptor CXCR4. These cells displayed cancer stem cell features, including increased self-renewal and differentiation ability in vitro and in vivo. They not only formed allografts containing immature bone regions but also disseminated into different organs, including lung, liver and bone. Experiments based on RNA interference revealed that Oct-4 expression drives both their engraftment and metastasis formation. This work points out the crucial contribution of Oct-4-expressing TICs in the hierarchical organization of the malignant potential, leading to metastasis formation. Consequently, it provides an appropriate model to develop novel therapies aiming to strike down TICs by targeting self-renewal genes, therefore efficient to reduce tumour growth and metastatic disease.

Improved Knockout Methodology Reveals That Frog Virus 3 Mutants Lacking either the 18K Immediate-Early Gene or the Truncated vIF-2 α Gene Are Defective for Replication and Growth In Vivo

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.

Efficient gene targeting mediated by a lentiviral vector-associated meganuclease

Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes.

Generation of immunocompatible cardiac myocytes for heart repair

Background Repair of myocardia with stem cell derived cardiomyocytes (SC-CM) is becoming increasingly realistic, although major obstacles still exist, including immunorejection of the grafts. Expression of MHC class I (MHC-I) molecules by grafted cells is the main reason for rejection. Downregulation of MHC molecules in SC-CM should reduce the immunogenicity of these cells. Results Cardiomyocytes were derived from mouse embryonic SC (CorAt) in which puromycin resistance gene was driven by a cardiac-specific promoter. CorAt formed beating cardiomyocytes and expressed cardiac specific proteins. The effect of inflammatory stimuli on the expression of MHC-I molecules was mimicked by IFN-gamma treatment. MHC-I expression was reduced by RNA interference against beta2-microglobulin (light chain) as measured by qPCR. Transduction of CorAt with anti-beta2-microglobulin shRNA lentivirus led to a lowered activation of cognate T-cells when MHC-I downregulated CorAt were used as targets. Similar results were obtained with W19 fibroblasts, confirming that analogous strategy can be used for a variety of cells. Conclusions The CorAT cells were effective in the repair of ischemic myocardia of syngeneic mice. Making these cells immunocompatible with allo- or xenogeneic recipients offers a reliable source of ESC-CM for small rodents and can be extended to ESC of large mammals for human heart repair.

Orderly Inactivation of the Key Checkpoint Protein Mitotic Arrest Deficient 2 (MAD2) during Mitotic Progression

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.

Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

Unique Primitive Erythropoiesis Defect In Rpl5-Deficient Murine Embryonic Stem Cell Model of Diamond Blackfan Anemia

AbstractAbstract 877 Background:Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods:We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results:We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel).For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion:Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Gene Transfer Approach towards Hemophilia A Correction Using the Piggybac Transposon Vector.

AbstractAbstract 1477Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBac transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008–15013, 2006). Advantages of this novel nonviral vector system include a large transgene cassette capacity and ease of production and purification. We hypothesize that a PB transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) or hyperactive (hyp-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PB transposons were engineered to carry a puromycin resistance gene (PB puro), a human alpha1 antitrypsin reporter (PB hAAT), or hFVIII gene (B domain deleted or a partial B domain-226 amino acids/N6). We evaluated co- and hyp-transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB puro vector, we demonstrated that the hyp-transposase generated a 2 fold higher transposition efficiency than the co-transposase in hepatocytes. We investigated the impact of varying the ratio of transposon to transposase; we screened ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the Huh-7 cell line. Overall, the 1:2 and 1:1 ratios gave the greatest transposition efficiency in vitro. We evaluated the in vivo gene transfer efficiency in mice by hydrodynamic tail-vein injection using PB hAAT driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Either a low (5 micrograms transposon) or high (25 micrograms transposon) dose was given with varying amounts of hyp-transposase to generate an in vivo dose response curve. Serum hAAT levels were measured prior to injection and then monthly for 3 months. Results revealed the 1:1 ratio at the high transposon dose generated higher level of expression compared to all other doses with expression stable in all groups for 3 months. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression of a secreted protein. Disclosures:Staber:Bayer Healthcare: Research Funding.

Long-Term Improvement in Postinfarct Left Ventricular Global and Regional Contractile Function Is Mediated by Embryonic Stem Cell–Derived Cardiomyocytes

Pluripotent stem cells represent one promising source for cellular cardiomyoplasty. In this study, we used cardiac magnetic resonance to examine the ability of highly enriched cardiomyocytes (CMs) derived from murine embryonic stem cells (ESC) to form grafts and improve contractile function of infarcted rat hearts. Highly enriched ESC-CMs were obtained by inducing cardiac differentiation of ESCs stably expressing a cardiac-restricted puromycin resistance gene. At the time of transplantation, enriched ESC-CMs expressed cardiac-specific markers and markers of developing CMs, but only 6% of them were proliferating. A growth factor-containing vehicle solution or ESC-CMs (5 to 10 million) suspended in the same solution was injected into athymic rat hearts 1 week after myocardial infarction. Initial infarct size was measured by cardiac magnetic resonance 1 day after myocardial infarction. Compared with vehicle treatment, treatment with ESC-CMs improved global systolic function 1 and 2 months after injection and significantly increased contractile function in initially infarcted areas and border zones. Immunohistochemistry confirmed successful engraftment and the persistence of α-actinin-positive ESC-CMs that also expressed α-smooth muscle actin. Connexin-43-positive sites were observed between grafted ESC-CMs but only rarely between grafted and host CMs. No teratomas were observed in any of the animals. Highly enriched and early-stage ESC-CMs were safe, formed stable grafts, and mediated a long-term recovery of global and regional myocardial contractile function after infarction.

Resveratrol Inhibits mTOR Signaling by Promoting the Interaction between mTOR and DEPTOR

Resveratrol (RSV) is a naturally occurring polyphenol that has been found to exert antioxidant, anti-inflammatory, and neuroprotective properties. However, how RSV exerts its beneficial health effects remains largely unknown. Here, we show that RSV inhibits insulin- and leucine-stimulated mTOR signaling in C2C12 fibroblasts via a Sirt1-independent mechanism. Treating C2C12 cells with RSV dramatically inhibited insulin-stimulated Akt, S6 kinase, and 4E-BP1 phosphorylation but had little effect on tyrosine phosphorylation of the insulin receptor and activation of the p44/42 MAPK signaling pathway. RSV treatment also partially blocked mTOR and S6 kinase phosphorylation in TSC1/2-deficient mouse embryonic fibroblasts, suggesting the presence of an inhibitory site downstream of TSC1/2. Knocking out PDK1 or suppressing AMP-activated protein kinase had little effect on leucine-stimulated mTOR signaling. On the other hand, RSV significantly increased the association between mTOR and its inhibitor, DEPTOR. Furthermore, the inhibitory effect of RSV on leucine-stimulated mTOR signaling was greatly reduced in cells in which the expression levels of DEPTOR were suppressed by RNAi. Taken together, our studies reveal that RSV inhibits leucine-stimulated mTORC1 activation by promoting mTOR/DEPTOR interaction and thus uncover a novel mechanism by which RSV negatively regulates mTOR activity.