Calcium Phosphate Coprecipitation Research Articles

Human ADA3 Binds to Estrogen Receptor (ER) and Functions As a Coactivator for ER-mediated Transactivation

We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target. Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation. Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily. We show that ADA3 directly interacts with ER alpha and ER beta. Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2. Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation. Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene. More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes. The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes. Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.

High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca–Mg phosphate

Transfer of desirable genetic sequences into mammalian cells is an essential tool for analysis of gene structure, functions and regulation and industry-based production of therapeutically important proteins and pivotal for gene therapy and DNA vaccination strategies. Considering some severe limitations of viral systems including immunogenicity, carcinogenicity and so on, synthetic nonviral systems are highly desirable in the above applications. However, existing nonviral techniques are extremely inefficient compared to the viral ones. Therefore, we report here on the development of a highly efficient synthetic device for gene delivery and expression into mammalian cells, based on controllable growth of nanoapatite particles. Mg 2+ incorporation into the apatite particles caused significant inhibition of particle growth, resulting in retention of nanosized particles which contributed remarkably to the cellular uptake of DNA and its subsequent expression (>10-fold) compared with classical calcium phosphate coprecipitation, one of the most widely used transfection methods.

Casein Kinase Iϵ Plays a Functional Role in the Transforming Growth Factor-β Signaling Pathway

The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.

893. Effect of Hepatic Insulin Gene Therapy on Intrahepatic Carbohydrate Metabolism In Vivo

Osteoarthiritis (OA) is a significant social and economic problem without adequate therapies. OA is characterized by focal erosion of articular cartilage in the joints of affected individuals. Recent advances in the field of gene therapy have identified potential transgenes that may alleviate symptoms and potentially regenerate the damaged intra-articular surfaces of the OA joint. Injecting adenovirus into the articular capsule results in preferential transduction of the synovium. Unfortunately, therapeutic growth factor expression from the synovial tissue results in ectopic cartilage formation and may cause osteophyte formation. One solution to overcome this is to express chondrogenic growth factors within the cartilage by transducing chondrocytes. However, poor transduction of articular chondrocytes limits efficient adenoviral mediated gene transfer strategies for the treatment of OA. To this end, we tested whether including adenovirus in a calcium phosphate co-precipitate (Ad:CaPi) would increase gene transfer efficiency in rat chondrosarcoma cells (RCS) in vitro. When cells were transduced with equivalent MOI, we found a significant increase in βgal expression (2-log) in Ad:CaPi transduced cells compared to adenovirus alone. Moreover, a dose response comparison between adenovirus and Ad:CaPi showed a significant increase in efficiency, where 1 log fewer particles of adenovirus in calcium phosphate complexes were required to transduce the same number of cells as adenovirus alone. Since Ad:CaPi complexes increase gene transfer to different cell types through a receptor independent mechanism, it is likely that these complexes will increase gene transfer to all cell types in the articular space including the surrounding synovial tissue, and therefore would require genetic modification to achieve chondrocyte specific gene expression. Therefore, we tested an alternative approach to re-direct adenoviral gene transfer specifically to chondrocytes. We utilized a biotinylated anti-α10 integrin antibody that is specific for chondrocytes. We coupled the antibody with a multivalent neutravidin, and complexed this with a metabolically biotinylated adenovirus. The results indicate a 5-fold increase in gene transfer with the antibody-targeted adenovirus compared to the biotinylated adenovirus alone. Importantly, since this virus has significantly reduced CAR binding, this increase may signify a selectable advantage for transducing chondrocytes within the joint capsule. From this data, we identify novel methods to increase gene transfer to chondrocytes in vitro, and indicate a potential local and chondrocyte targeted gene therapy strategy in vivo.

66. A safe and efficient lentiviral vector system: optimisation of virus production and purification

Top of pageAbstract We have previously described 5 and 6 plasmid systems for production of recombinant, HIV-1 derived, lentiviral vectors (Fuller and Anson, 20011. These systems utilise codon-optimised reading frames for expression of Gag/GagPol. We have now further developed these systems by constructing more efficient expression constructs, and by utilising codon-optimized reading frames for all HIV-1 proteins (with the exception of Env, the virus being pseudotyped with VSV-G). In addition, we have refined the design of our vector construct to increase the synthesis of genomic transcripts. By careful titration of plasmid ratios and absolute amounts, and by careful optimization of the conditions for transfection of 293T cells and for virus collection, we have increased the titre of virus that can be produced with the 5-plasmid system over 8-fold. We can now routinely produce virus using the 5-plasmid system with a titre of >5 × 107 A549 transducing units per ml. Vectors using a liver specific (alpha-1-antitrypsin gene) promoter, and two different ubiquitous (phosphoglycerate kinase gene and myeloproliferative sarcoma virus long terminal repeat) promoters, for transgene expression have been constructed. All three vectors are available with either the enhanced yellow fluorescent protein or LacZ marker genes. To further increase the safety of our vector system we have manipulated the codon usage in the GagPol expression (helper) construct to minimize homology with the HIV-1 gag gene sequence that is present in our vector constructs. We show that this results in a significant decrease in the transfer of gagpol gene sequences to transduced cells without reducing the efficiency of virus production. Large-scale production of virus was facilitated by using a serum free medium (OptiPro-SFM, Invitrogen) for virus collection which allowed rapid and efficient processing of viral supernatant using a combination of ultrafiltration and ultracentrifugation. OptiPro-SFM was superior to a number of other serum free media that were tested. The use of coated cell culture vessels, sodium butyrate, or BES in place of HeBS for calcium phosphate coprecipitation, did not improve viral titres. In conclusion, we now have an extremely safe and efficient recombinant HIV-1 vector system available with a choice of marker genes and internal promoters.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish.

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Is calcium oxalate nucleation in postprandial urine of males with idiopathic recurrent calcium urolithiasis related to calcium phosphate nucleation and the intensity of stone formation? Studies allowing insight into a possible role of urinary free citrate and protein.

In idiopathic recurrent urolithiasis (IRCU) calcium oxalate and calcium phosphate are components of stones. It is not sufficiently known whether in urine the nucleation (liquid-solid transition) of each salt requires a different environment, if so which environment, and whether there is an impact on stone formation. Nucleation was induced by in vitro addition of oxalate or calcium to post-test meal load whole urine of male stone patients (n=48), showing normal daily and baseline fasting oxaluria. The maximally tolerated (until visible precipitates occur) concentration of oxalate (T-Ox) or calcium (T-Ca) was determined; additionally evaluated were other variables in urine, including total, complexed and free citrate (F-Cit), protein (albumin, non-albumin protein) and the clinical intensity (synonymous metabolic activity; MA) of IRCU. In the first of three trials the accumulation of substances in stone-forming urine was verified (trial-V); in the second (clinical trial 1) two strata of T-Ox (Low, High) were compared; in the third (clinical trial 2) IRCU patients (n=27) and a control group (n=13) were included to clarify whether in stone-forming urine the first crystal formed was calcium oxalate or calcium phosphate, and to identify the state of F-Cit. T-Ox was studied at the original pH (average < 6.0), T-Ca at prefixed pH 6.0; the precipitates were subjected to electron microscopy and element analysis. Trial-V: Among the urinary substances accumulating at the indicated pHs were calcium, oxalate and phosphate, and the crystal-urine ratios were compatible with the nucleation of calcium oxalate, calcium-poor and calcium-rich calcium phosphate; citrate, protein and potassium also accumulated. Clinical trial 1: the two strata exhibited an inverse change of T-Ox and T-Ca, the ratio T-Ox/T-Ca and MA. The initial (before induction of Ox or Ca excess) supersaturation of calcium oxalate and brushite were unchanged, with the difference of proteinuria being borderline. Several correlations were significant (p < or = 0.05): urine pH with citrate and volume, protein with volume and MA, T-Ox with T-Ca and MA. Clinical trial 2: in patients with reduced urine volume and moderate urine calcium excess, the first precipitate appeared to be calcium oxalate, followed by amorphous calcium phosphate. Conversely, when the calcium excess was extreme, calcium-rich hydroxyapatite developed, followed by calcium oxalate; F-Cit, not total and complexed citrate, was decreased in IRCU vs. male controls; F-Cit rose pH-dependently, and the ratio F-Cit at original pH vs. F-Cit at pH 6.0 correlated inversely with the nucleation index T-Ox/T-Ca; MA correlated inversely with the ratio F-Cit at pH 6.0, respectively, original pH, but directly with the urinary albumin/non-albumin protein ratio. In summary 1) to study calcium oxalate and calcium phosphate nucleation in whole urine of IRCU patients is feasible; 2) at this crystallization stage the two substances, dominant in calcium stones, appear intimately linked, 3) in stone-forming urine, calcium phosphate may be ubiquitously present, likely as particles < 0.22 microm; 4) together with co-precipitation of calcium oxalate and calcium phosphate, low F-Cit and alteration of proteinuria may act in concert and accelerate stones.

Calfection: a novel gene transfer method for suspension cells

We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5×10 5 cells/ml in the presence of 9 mM calcium and 5 μg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to −80 °C. In contrast, calcium phosphate–DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium–plasmid DNA mixture through a 0.2-μm filter did not compromise protein expression, whereas calcium phosphate–DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates.

Development of a calcium phosphate co-precipitate/poly(lactide- co-glycolide) DNA delivery system: release kinetics and cellular transfection studies

One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide- co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

Reciprocal Inhibition between MyoD and STAT3 in the Regulation of Growth and Differentiation of Myoblasts

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.

Characterization of an Osteoblast-specific Enhancer Element in the CBFA1 Gene

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.

Regulation of Lipoprotein Lipase by the Oxysterol Receptors, LXRα and LXRβ

Lipoprotein lipase (LPL) is a key enzyme for lipoprotein metabolism and is responsible for hydrolysis of triglycerides in circulating lipoproteins, releasing free fatty acids to peripheral tissues. In liver, LPL is also believed to promote uptake of high density lipoprotein (HDL)-cholesterol and thereby facilitate reverse cholesterol transport. In this study we show that the Lpl gene is a direct target of the oxysterol liver X receptor, LXRalpha. Mice fed diets containing high cholesterol or an LXR-selective agonist exhibited a significant increase in LPL expression in the liver and macrophages, but not in other tissues (e.g. adipose and muscle). Studies in Lxr-deficient mice confirmed that this response was dependent more on the presence of LXRalpha than LXRbeta. Analysis of the Lpl gene revealed the presence of a functional DR4 LXR response element in the intronic region between exons 1 and 2. This response element directly binds rexinoid receptor (RXR)/LXR heterodimers and is sufficient for rexinoid- and LXR agonist-induced transcription of the Lpl gene. Together, these studies further distinguish the roles of LXRalpha and beta and support a growing body of evidence that LXRs function as key regulators of lipid metabolism and are anti-atherogenic.

Nkx2-5 activity is essential for cardiomyogenesis.

The homeobox transcription factor tinman is essential for heart vessel formation in Drosophila. In contrast, mice lacking the murine homologue Nkx2-5 are defective in cardiac looping but not in cardiac myocyte development. The lack of an essential role for Nkx2-5 in cardiomyogenesis in mammalian systems is most likely the result of genetic redundancy with family members. In this study, we used a dominant negative mutant of Nkx2-5, created by fusing the repressor domain of engrailed 2 to the Nkx2-5 homeodomain, termed Nkx/EnR. Expression of Nkx/EnR inhibited Me(2)SO-induced cardiomyogenesis in P19 cells but not skeletal myogenesis. Nkx/EnR inhibited expression of cardiomyoblast markers, such as GATA-4 and MEF2C, but not of mesoderm markers, such as Brachyury T and Wnt5b, or of skeletal lineage markers, such as MyoD and Mox1. To identify the minimal region of Nkx2-5 that can trigger cardiomyogenesis, we analyzed the activity of various Nkx2-5 deletion mutants. The C-terminal domain was not necessary for the ability of Nkx2-5 to induce cardiomyogenesis and loss of this domain did not enhance myogenesis. Therefore, Nkx2-5 function is essential for commitment of mesoderm into the cardiac muscle lineage, and the N-terminal region, together with the homeodomain, is sufficient for cardiomyogenesis in P19 cells.

Growth Hormone Regulates Phosphorylation and Function of CCAAT/Enhancer-binding Protein β by Modulating Akt and Glycogen Synthase Kinase-3

Growth hormone (GH) regulates transcription factors associated with c-fos, including C/EBPbeta. Two forms of C/EBPbeta, liver-activating protein (LAP) and liver inhibitory protein (LIP), are dephosphorylated in GH-treated 3T3-F442A fibroblasts. GH-induced dephosphorylation of LAP and LIP is reduced when cells are preincubated with phosphatidylinositol 3'-kinase (PI3K) inhibitors. GH activates Akt and inhibits glycogen synthase kinase-3 (GSK-3). Lithium, a GSK-3 inhibitor, increases GH-dependent dephosphorylation of LAP and LIP. Both are in vitro substrates of GSK-3, suggesting that GSK-3 inactivation contributes to GH-promoted dephosphorylation of C/EBPbeta. Alkaline phosphatase increases binding of LAP homodimers and decreases binding of LIP homodimers to c-fos, suggesting that dephosphorylation of C/EBPbeta modifies their ability to bind DNA. Both alkaline phosphatase- and GH-mediated dephosphorylation comparably increase binding of endogenous LAP in 3T3-F442A cells. In cells overexpressing LAP and GSK-3, LAP binding decreases, suggesting that GSK-3-mediated phosphorylation interferes with LAP binding. Expression of constitutively active GSK-3 reduced GH-stimulated c-fos promoter activity. These studies indicate that PI3K/Akt/GSK-3 mediates signaling between GH receptor and the nucleus, promoting dephosphorylation of C/EBPbeta. Dephosphorylation increases binding of LAP complexes to the c-fos promoter and may contribute to the participation of C/EBPbeta in GH-stimulated c-fos expression.

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells.

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

Cbfa1 Contributes to the Osteoblast-specific Expression of type I collagen Genes

Type I collagen is composed of two chains, alpha1(I) and alpha2(I), encoded by two distinct genes, the alpha1(I) and alpha2(I) collagen genes, that are highly expressed in osteoblasts. In most physiological situations, alpha1(I) and alpha2(I) collagen expression is coregulated, suggesting that identical transcription factors control their expression. Here, we studied the role of Cbfa1, an osteoblast-specific transcription factor, in the control of alpha1(I) and alpha2(I) collagen expression in osteoblasts. A consensus Cbfa1-binding site, termed OSE2, is present at the same location in the alpha1(I) collagen promoter at approximately -1347 base pairs (bp) of the rat, mouse, and human genes. Cbfa1 can bind to this site, as demonstrated by electrophoretic mobility shift assay (EMSA) and supershift experiments using an anti-Cbfa1 antibody. Mutagenesis of the alpha1(I) collagen OSE2 at -1347 bp reduced the activity of a alpha1(I) collagen promoter fragment 2- to 3-fold. Moreover, multimers of this OSE2 at -1347bp confer osteoblast-specific activity to a minimum alpha1(I) collagen promoter fragment in DNA transfection experiments as well as in transgenic mice. An additional Cbfa1-binding element is present in the alpha1(I) collagen promoter of mouse, rat, and human at approximately position -372. This site binds Cbfa1 only weakly and does not act as a cis-acting activator of transcription when tested in DNA transfection experiments. Similar to alpha1(I) collagen, the mouse alpha2(I) collagen gene contains multiple OSE2 sites, of which one is conserved across multiple species. In EMSA, Cbfa1 binds to this site and multimers of this alpha2(I) OSE2 element confer osteoblast-specific activity to the minimum alpha1(I) collagen promoter in DNA transfection experiments. Thus, our results suggest that Cbfa1 is one of the positive regulators of the osteoblast-specific expression of both type I collagen genes.

Biomimetic coprecipitation of calcium phosphate and bovine serum albumin on titanium alloy.

Titanium alloy implants were precoated biomimetically with a thin and dense layer of calcium phosphate and then incubated either in a supersaturated solution of calcium phosphate or in phosphate-buffered saline, each containing bovine serum albumin (BSA) at various concentrations, under physiological conditions for 48 h. Coated implants then underwent scanning electron microscopy, immunohistochemical evaluation, Fourier transform infrared spectroscopy, and X-ray diffraction. The quantity of BSA taken up by coatings and the kinetics of protein release were monitored colorimetrically. In coatings prepared by the coprecipitation of calcium phosphate and BSA, protein had become incorporated into the mineral crystal latticework. With increasing BSA concentration, matrices decreased in thickness, became more dense, showed lower crystallinity, and underwent a change in crystal geometry. The octacalcium phosphate structure manifested in the absence of protein was gradually transformed into a carbonated apatite form. Preformed mineral coatings became only superficially mantled with a layer of BSA, and the morphology of the mineral matrices themselves remained unchanged. At equivalent protein concentrations, coatings prepared by the coprecipitation of calcium phosphate released only a minute fraction of its protein component under physiological conditions, whereas preformed mineral matrices showed a "burst" release of their associated protein within a single 2-h period. The biomimetic coating can be a carrier for osteoinductive agents.

Concentration of viral vectors by co-precipitation with calcium phosphate.

The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks. The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors. Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50-100% recovery, and titers can be concentrated up to 1,000-fold. The calcium phosphate co-precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co-precipitation can be carried out if required.

Inhibition of vascular endothelial growth factor activity by transfection with the soluble FLT-1 gene.

Vascular endothelial growth factor (VEGF)-induced angiogenesis is involved in the etiology of some cardiovascular diseases. The soluble form of VEGF receptor, FLT-1 (sFLT-1), is a potent antagonist of VEGF. Therefore, we investigated whether transfection with the sFLT-1 gene could inhibit VEGF-induced angiogenesis. Human embryonic kidney (HEK)-293 cells were transfected with plasmids containing VEGF and sFLT-1 (pCMV-VEGF and pCMV-sFLT-1) by the calcium-phosphate co-precipitation method. VEGF- and/or sFLT-1-transfected HEK-293 cells were incubated for 24 h, and then conditioned medium was collected. The effects of conditioned medium on angiogenesis were tested by incorporation of [3H]thymidine into human umbilical vein endothelial cells (HUVECs). Expression of VEGF protein was determined by Western blotting. The conditioned medium from sFLT-1 gene-transfected HEK-293 cells significantly inhibited recombinant VEGF-induced increase in [3H]thymidine incorporation by HUVECs. VEGF gene-transfected HEK-293 cells secreted VEGF protein into conditioned medium. This conditioned medium increased [3H]thymidine incorporation by HUVECs, which was significantly inhibited by co-transfection of sFLT-1 gene with VEGF gene. These observations suggested that sFLT-1 gene transfer could inhibit VEGF-induced DNA synthesis of vascular endothelial cells.